Antibody-immobilized carrier, method of producing antibody-immobilized carrier, and use of said antibody-immobilized carrier

ABSTRACT

The present invention provides an antibody-immobilized carrier that can be used in antibody screening, a method of producing the antibody-immobilized carrier, and use of the antibody-immobilized carrier. Efficient antibody screening can be carried out particularly by an antibody-immobilized carrier including two or more antibody immobilized regions onto each of which a heavy-chain low-molecular-weight antibody and a light-chain low-molecular-weight antibody are separately immobilized, the two or more antibody immobilized regions each being included in an independent manner, the heavy-chain low-molecular-weight antibody including a heavy-chain variable region, the light-chain low-molecular-weight antibody including a light-chain variable region, the heavy-chain low-molecular-weight antibody and the light-chain low-molecular-weight antibody each being derived from an antibody recognizing a different antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Section 371 U.S. national stage entry ofpending International Patent Application No. PCT/JP2011/053157,International Filing Date Feb. 15, 2011, which published on Aug. 25,2011 as Publication No. WO 2011/102342, which claims the benefit ofJapanese Patent Application No. 2010-031684, filed Feb. 16, 2010, thecontents of which are incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to an antibody-immobilized carrier,particularly to a carrier onto which lower molecular weight antibodiesare immobilized, a method of producing the antibody-immobilized carrier,and use of the antibody-immobilized carrier.

BACKGROUND ART

Conventionally, immunoassay in which a trace substance is detected byutilizing antigen-antibody reaction has been known. Moreover, recently,antibody medicines utilizing functions of antibodies have been activelydeveloped. Such antibody medicines have excellent effects and lessadverse effects, and act on various drug targets. Moreover, the antibodymedicines can be industrially produced. Accordingly, the antibodymedicines are drawing attentions, as a technology that allows quicklyproviding a treatment against target molecules found in genomeresearches. The antibody medicines have various functions of, forexample, a blocking antibody that is combined to a receptor or a ligandand inhibiting signaling, a signaling antibody that is combined to areceptor and shows a receptor crosslinking effect, and a targetingantibody having ADCC activity or CDC activity and therefore havingcytotoxicity.

Development of such antibody medicines generally starts with gene searchfollowed by identification of an antigen that becomes a target of anantibody medicine, preparation of an antibody that binds specifically tothe antigen, check of a pharmacological effect of the antibody, andultimate mass production of the antibody medicine. In this developmentflow, a process of screening an antibody that binds specifically to anantigen is considered important.

As a technique for screening antibodies, a method employing phagedisplay is known. This method is a technique in which: first, anantibody library presenting various antigen-specific antibodies onphages is prepared; and then, screening of antibodies that bindsspecifically to a specific antigen is carried out (See Patent Literature1, for example).

CITATION LIST Patent Literature

-   Patent Literature 1-   Publication of Japanese Translation of PCT International    Application, Tokuhyou, No. 2009-511892 A (Publication Date: Mar. 19,    2009)

SUMMARY OF INVENTION Technical Problem

However, an antibody screening method utilizing the phage displaydescribed above is not sufficient. This is because: (a) work operationis complicated; (b) isolation of a positive clone is difficult; and (c)moreover, false positive clones may be selected. Therefore, developmentof a novel technique, other than the phage display, that can be used inantibody screening has been strongly desired.

The present invention is attained in view of the above problems. Anobject of the present invention is to provide an antibody-immobilizedcarrier which can be used in antibody screening, a method of producingthe antibody-immobilized carrier, and use of antibody-immobilizedcarrier.

Solution to Problem

As a result of diligent studies for achieving the object above, theinventors of the present invention developed an innovative techniquethat made it possible to carry out effective screening of an antibodyrecognizing a specific antigen. In this technique, first, heavy chainsand light chains derived from different antibodies are made into smallermolecules, and then a number of combinations of the smaller moleculesare immobilized onto a carrier. As a result, the inventors accomplishedthe present invention. In other words, the present invention isconfigured as follows:

(1) An antibody-immobilized carrier including at least one antibodyimmobilized region where a heavy-chain low-molecular-weight antibody anda light-chain low-molecular-weight antibody are separately immobilized,the at least one antibody immobilized region being included in anindependent manner, the heavy-chain low-molecular-weight antibodyincluding a heavy-chain variable region, the light-chainlow-molecular-weight antibody including a light-chain variable region,the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody each being derived from an antibodyrecognizing a different antigen.

(2) The antibody-immobilized carrier as set forth in (1), wherein: eachof the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody is separately immobilized onto a carriervia a carrier binding peptide that binds to a material of a carriersurface; and the carrier binding peptide is provided to a C-terminusside of the heavy-chain variable region in the heavy-chainlow-molecular-weight antibody or to a C-terminus side of the light-chainvariable region in the light-chain low-molecular-weight antibody.

(3) The antibody-immobilized carrier as set forth in (2), wherein: thematerial of the carrier surface is plastic resin having beenhydrophilized by property modification, the plastic resin beingpolystyrene, polycarbonate, polypropylene, polyethylene,polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA).

(4) The antibody-immobilized carrier as set forth in (2) or (3),wherein: the carrier binding peptide is a peptide that binds tohydrophilic polystyrene, hydrophilic polycarbonate, hydrophilicpolypropylene, hydrophilic polyethylene, hydrophilicpolydimethylsiloxane (PDMS) or hydrophilic polymethyl methacrylate(PMMA).

(5) The antibody-immobilized carrier as set forth in any one of (1) to(4), wherein: the heavy-chain low-molecular-weight antibody is aheavy-chain low-molecular-weight antibody consisting of the heavy-chainvariable region or a heavy-chain low-molecular-weight antibody (Fab H)consisting of a heavy-chain variable region and a first heavy-chainconstant region (CH₁).

(6) The antibody-immobilized carrier as set forth in any one of (1) to(5), wherein: the light-chain low-molecular-weight antibody is alight-chain low-molecular-weight antibody consisting of the light-chainvariable region or a light-chain low-molecular-weight antibody (Fab L)consisting of a light-chain variable region and a light-chain constantregion (C_(k)).

(7) A method of producing an antibody-immobilized carrier including thestep of: immobilizing a heavy-chain low-molecular-weight antibodyincluding a heavy-chain variable region and a light-chainlow-molecular-weight antibody including a light-chain variable regionseparately onto a carrier, so that an antibody immobilized region isprepared, the heavy-chain low-molecular-weight antibody and thelight-chain low-molecular-weight antibody each being derived from anantibody recognizing a different antigen.

(8) The method as set forth in (7), wherein the step of immobilizing isrepeated at least two times so that two or more of the antibodyimmobilized region are provided in an independent manner.

(9) The method as set forth in (7), wherein the step of immobilizingincluding: (a) the first sub-step of immobilizing, onto the carrier,insoluble aggregates of the heavy-chain low-molecular-weight antibodyand the light-chain low-molecular-weight antibody by putting thedenatured aggregates in contact with a carrier surface, the denaturedinsoluble aggregates each having been denatured by a denaturing agentand being in a denatured state; and (b) the second sub-step of refoldingthe heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody each in the denatured state, by removingthe denaturing agent from the heavy-chain low-molecular-weight antibodyand the light-chain low-molecular-weight antibody that are in thedenatured state and immobilized.

(10) The method as set forth in (9), wherein the first sub-step and thesecond sub-step in the step of immobilizing are carried out, separatelyfor each of the heavy-chain low-molecular-weight antibody and thelight-chain low-molecular-weight antibody.

(11) The method as set forth in (10), wherein: in the step ofimmobilizing, after the first sub-step and the second sub-step arecarried out for the light-chain low-molecular-weight antibody, the firstsub-step and the second sub-step are carried out for the heavy-chainlow-molecular-weight antibody.

(12) The method as set forth in any one of (9) to (11), wherein: thefirst sub-step employs, as the denaturing agent, urea whoseconcentration is in a range of 0.5 M to 4M.

(13) The antibody-immobilized carrier obtained by the method as setforth in any one of (8) to (12) above.

(14) An antibody screening method including the step of: screening aheavy-chain low-molecular-weight antibody and/or a light-chainlow-molecular-weight antibody each recognizing a specific antigen, byusing the antibody-immobilized carrier as set forth in any one of (1) to(6) and (13) above.

(15) A method of screening a human antibody recognizing a specificantigen by using the antibody-immobilized carrier as set forth in anyone of (1) to (6), and (13) above, the screening being carried out byusing a chimeric antibody or a humanized antibody each recognizing thespecific antigen, the heavy-chain low-molecular-weight antibody beingimmobilized onto the at least one antibody immobilized region andincluding a heavy-chain variable region derived from the chimericantibody or the humanized antibody, the light-chain low-molecular-weightantibody being a light-chain low-molecular-weight antibody that isimmobilized onto the at least one antibody immobilized region and thatincludes a light-chain variable region derived from a random humanantibody, the method including the steps of: (i) putting the specificantigen in contact with the antibody-immobilized carrier; (ii) detectingan antibody immobilized region recognizing the specific antigen on theantibody-immobilized carrier; and (iii) determining a light-chainlow-molecular-weight antibody immobilized on the antibody immobilizedregion detected in the step (ii), as a candidate for a light-chainvariable region of the human antibody recognizing the specific antigen.

(16) The method of screening as set forth in (15), further including thesteps of: (iv) putting the specific antigen in contact with anotherantibody-immobilized carrier including another antibody immobilizedregion onto which (a) the light-chain low-molecular-weight antibodydetermined as the candidate in the step (iii) and (b) a heavy-chainlow-molecular-weight antibody including a heavy-chain variable regionderived from a random human antibody are immobilized; (v) detecting anantibody immobilized region recognizing the specific antibody on theanother antibody-immobilized carrier; and (vi) determining a heavy-chainlow-molecular-weight antibody immobilized onto the antibody immobilizedregion detected in the step (v), as a candidate for a heavy-chainvariable region of the human antibody recognizing the specific antigen.

(17) A method of screening a human antibody recognizing a specificantigen by using the antibody-immobilized carrier as set forth in anyone of (1) to (6), and (13) above, the screening being carried out byusing a chimeric antibody or a humanized antibody each recognizing thespecific antigen, the light-chain low-molecular-weight antibody beingimmobilized onto the at least one antibody immobilized region andincluding a light-chain variable region derived from the chimericantibody or the humanized antibody, the heavy-chain low-molecular-weightantibody being a heavy-chain low-molecular-weight antibody that isimmobilized onto the at least one antibody immobilized region and thatincludes a heavy-chain variable region derived from a random humanantibody, the method including the steps of: (i) putting the specificantigen in contact with the antibody-immobilized carrier; (ii) detectingan antibody immobilized region recognizing the specific antigen on theantibody-immobilized carrier; and (iii) determining a heavy-chainlow-molecular-weight antibody immobilized on the antibody immobilizedregion detected in the step (ii), as a candidate for a heavy chainvariable region of the human antibody recognizing the specific antigen.

(18) The method of screening as set forth in (17), further including thesteps of: (iv) putting the specific antigen in contact with anotherantibody-immobilized carrier including another antibody immobilizedregion onto which (a) the heavy-chain low-molecular-weight antibodydetermined as the candidate in the step (iii) and (b) a light-chainlow-molecular-weight antibody including a light-chain variable regionderived from a random human antibody are immobilized; (v) detecting anantibody immobilized region recognizing the specific antibody on theanother antibody-immobilized carrier; and (vi) determining a light-chainlow-molecular-weight antibody immobilized onto the antibody immobilizedregion detected in the step (v), as a candidate for a light-chainvariable region of the human antibody recognizing the specific antigen.

(19) A method of producing a human antibody, the method including thestep of producing the human antibody by combining the candidate for thelight-chain variable region of the human antibody and the candidate forthe heavy-chain variable region of the human antibody, the candidate forthe light-chain variable region and the candidate for the heavy-chainvariable region being determined by the method of screening as set forthin any one of (15) to (18) above.

Advantageous Effects of Invention

Because the antibody-immobilized carrier of the present invention is acarrier onto which a number of combinations of a heavy chain and a lightchain each derived from a different antibody are immobilized, screeningof antibodies recognizing a specific antigen can be efficiently carriedout. The antibody-immobilized carrier is applicable not only to theabove but also to screening of antibodies for diagnosis. Further,antibody-immobilized carrier is also applicable to various immunoassaysutilizing antigen-antibody reaction.

For a fuller understanding of the nature and advantages of theinvention, reference should be made to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

(a) of FIG. 1 is a diagram schematically showing a state where a set ofa heavy-chain low-molecular-weight antibody and a light-chainlow-molecular-weight antibody each immobilized onto a carrier reactswith an antigen; and (b) of FIG. 1 is a diagram schematically showing aconfiguration of an antibody-immobilized carrier onto which a number ofcombinations of a heavy-chain low-molecular-weight antibody and alight-chain low-molecular-weight antibody are immobilized.

FIG. 2 is a diagram showing a result of examination on antigen bindingactivity by using a carrier prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom mouse anti-RNase antibody: Fab H, Fab L, a low-molecular-weightantibody in which PS-tag is bound to Fab H, and a low-molecular-weightantibody in which PS-tag is bound to Fab L.

FIG. 3 is a diagram showing a result of examination on conditions forsolid phase refolding by using a carrier prepared by immobilizingthereon one or a combination of the following low-molecular-weightantibodies derived from mouse anti-RNase antibody: Fab H, Fab L, alow-molecular-weight antibody in which PS-tag is bound to Fab H, and alow-molecular-weight antibody in which PS-tag is bound to Fab L.

FIG. 4 is a diagram showing a result of detecting mouse RNase as anantigen by using a carrier prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom a mouse anti-CRP antibody, a mouse anti-RNase antibody, a humananti-IFNG antibody, and a human anti-ED-B antibody: (a)low-molecular-weight antibodies in each of which PS-tag is bound to FabH; and (b) low-molecular-weight antibodies in each of which PS-tag isbound to Fab L.

FIG. 5 is a diagram showing a result of detecting mouse CRP as anantigen by using a carrier prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom a mouse anti-CRP antibody, a mouse anti-RNase antibody, a humananti-IFNG antibody, and a human anti-ED-B antibody: (a)low-molecular-weight antibodies in each of which PS-tag is bound to FabH; and (b) low-molecular-weight antibodies in each of which PS-tag isbound to Fab L.

FIG. 6 is a diagram showing a result of detecting human ED-B as anantigen by using a carrier prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom a mouse anti-CRP antibody, a mouse anti-RNase antibody, a humananti-IFNG antibody, and a human anti-ED-B antibody: (a)low-molecular-weight antibodies in each of which PS-tag is bound to FabH; and (b) low-molecular-weight antibodies in each of which PS-tag isbound to Fab L.

FIG. 7 is a diagram showing a result of detecting human IFNG as anantigen by using a carrier prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom a mouse anti-CRP antibody, a mouse anti-RNase antibody, a humananti-IFNG antibody, and a human anti-ED-B antibody: (a)low-molecular-weight antibodies in each of which PS-tag is bound to FabH; and (b) low-molecular-weight antibodies in each of which PS-tag isbound to Fab L.

FIG. 8 is a diagram showing a result of examination on difference inantigen binding activity caused by difference in immobilization method,by using carriers carrier each prepared by immobilizing thereon one or acombination of the following low-molecular-weight antibodies derivedfrom mouse anti-RNase antibody: Fab H, Fab L, a low-molecular-weightantibody in which PS-tag is bound to Fab H, and a low-molecular-weightantibody in which PS-tag is bound to Fab L.

FIG. 9 is a diagram showing a result of examination on top 40 cloneswhich have higher affinity for AFP and which are selected from among amouse Fab H-PS library (960 types), in regard to affinity for an antigen(light emission intensity).

FIG. 10 is a diagram showing a result of examination on top 30 cloneswhich have higher affinity for AFP and which are selected from a mouseFab L-PS library (960 types), in regard to affinity for an antigen.

FIG. 11 is a diagram showing a result of examination on combinations ofan H chain derived from a chimeric antibody and an L chain selected froma human L chain library (960 types), in regard to affinity for anantigen.

FIG. 12 is a diagram showing a result of examination on top 95 clonesfrom among combinations of an H chain derived from a chimeric antibodyand an L chain selected from a human L chain library (960 types), inregard to affinity for an antigen.

FIG. 13 is a diagram showing a result of examination on combinations ofan L chain derived from a chimeric antibody and an H chain selected froma human H chain library (960 types), in regard to affinity for anantigen.

FIG. 14 is a diagram showing a result of examination on top 94 clonesfrom among combinations of an L chain derived from a chimeric antibodyand an H chain selected from a human H chain library (960 types), inregard to affinity for an antigen.

FIG. 15 is a diagram showing a result of examination on combinations (40types) of a human L chain clone and a human H chain clone, in regard toaffinity for an antigen.

FIG. 16 is a diagram showing a result of examination on combinations (40types) of a human L chain clone and a human H chain clone, in regard toaffinity for an antigen (relative activity; activity per stabilized Fabunit).

FIG. 17 is a diagram showing (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab L-PS librarywith Clone No. 1 of human Fab H-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab L-PSlibrary with Clone No. 2 of human Fab H-PS.

FIG. 18 is a diagram showing (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab L-PS librarywith Clone No. 3 of human Fab H-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab L-PSlibrary with Clone No. 4 of human Fab H-PS.

FIG. 19 is a diagram showing a result of Fab antibodies obtained bycombining 960 types in a human Fab L-PS library with Clone No. 11 ofhuman Fab H-PS.

FIG. 20 is a diagram showing (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab H-PS librarywith Clone No. 1 of human Fab L-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab H-PSlibrary with Clone No. 2 of human Fab L-PS.

FIG. 21 is a diagram showing (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab H-PS librarywith Clone No. 3 of human Fab L-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab H-PSlibrary with Clone No. 11 of human Fab L-PS.

FIG. 22 is a diagram showing a result of Fab antibodies obtained bycombining 960 types in a human Fab H-PS library with Clone No. 12 ofhuman Fab L-PS.

DESCRIPTION OF EMBODIMENTS

One embodiment of the present invention is described below. Note thatall professional literatures and patent literatures cited in the presentspecification are incorporated herein as references in the presentspecification. In the present specification, unless specifically noted,“A to B” indicating a range of numerical values means “A or more (i.e.,containing A and greater than A) and B or less (i.e., containing B andless than B). The wordings of all of “recognize an antigen”, “haveaffinity for an antigen” and “bind to an antigen” mean that an antibodycomponent immunologically reacts with an antigen. The above wordings aresynonyms and interchangeable with each other.

<1. Antibody-Immobilized Carrier>

An antibody-immobilized carrier of the present invention only needs toinclude at least one antibody immobilized region where a heavy-chainlow-molecular-weight antibody and a light-chain low-molecular-weightantibody are separately immobilized, the at least one antibodyimmobilized region being included in an independent manner, theheavy-chain low-molecular-weight antibody including at least aheavy-chain variable region, the light-chain low-molecular-weightantibody including at least a light-chain variable region, theheavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody each being derived from an antibodyrecognizing a different antigen. The antibody-immobilized carrier is notspecifically limited in regard to other configurations such as concretestructures, materials, and forms.

The “low-molecular-weight antibody” as used in the present specificationis an antibody fragment of a whole antibody (e.g., whole IgG) from whicha part is deficient and includes at least a heavy-chain variable region(VH) or light-chain variable region (VL). The “low-molecular-weightantibody” only needs to have an ability to bind to an antigen.Specifically preferable examples of the low-molecular-weight antibodyare VH, VL, and Fab, Fab′, and F(ab′)2 in a heavy chain or a lightchain. In particular, the heavy-chain low-molecular-weight antibody ispreferably a heavy-chain low-molecular-weight antibody consisting of (a)a heavy-chain variable region or (b) Fab H: a heavy-chain variableregion and a first heavy-chain constant region (CH₁). Meanwhile,particularly, the light-chain low-molecular-weight antibody ispreferably a light-chain low-molecular-weight antibody consisting of (a)a light-chain variable region or (b) Fab L: a light-chain variableregion and a light-chain constant region (C_(k)). The abovelow-molecular-weight antibodies are preferable because theselow-molecular-weight antibodies can be efficiently immobilized to acarrier.

For obtaining such a low-molecular-weight antibody, a conventionallyknown cloning technique or a conventionally known chemical synthesismethod can be used in production. For example, by employing the cloningtechnique, it is possible to collect a peptide having the amino acidsequence of the low-molecular-weight antibody by (i) first, preparingDNA encoding the antibody fragment (low-molecular-weight antibody), (ii)obtaining recombinant DNA, by inserting thus prepared DNA into anautonomously replicating vector, (iii) introducing as appropriate thusobtained recombinant DNA into a host such as E. coli, Bacillus subtilis,mycobacterium, yeast, filamentous fungi, a plant cell, an insect cell,an animal cell or the like and thereby obtaining a transformant, and(iv) culturing the transformant and collecting the peptide from thuscultured material (See, for example, Co, M. S. et al., J. Immunol.(1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol.(1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol.(1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; and Bird, R.E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

Alternatively, the low-molecular-weight antibody can be obtained by (i)preparing DNA encoding the low-molecular-weight antibody and (ii)synthesizing in an acellular protein synthesis system by using cellextract liquid of wheat germ or E. coli, or the like. As anotheralternative, the low-molecular-weight antibody can also be obtained bysuccessively performing dehydration synthesis and extension of an aminoacid with use of a common peptide chemical synthesis method such as a“solid-phase synthesis method” or a “liquid-phase synthesis method”.

In particular, an antibody production technique employing generecombination makes it possible to mass produce antibodies at low cost.Therefore, preferably the low-molecular-weight antibodies each having,as a basic skeleton, a variable region that binds to an antigen, areproduced by (i) first isolating an antibody producing gene from ageneral antibody producing cell strain, (ii) then preparing, from theantibody producing gene, a nucleotide sequence corresponding to thelow-molecular-weight antibody, and (iii) subsequently incorporating thenucleotide sequence into E. coli or the like.

The following provides an example of a method of producing thelow-molecular-weight antibody. First, from a hybridoma that produces anantibody, mRNA or total RNA encoding a variable region is isolated.Here, mRNA or total RNA may be isolated by a known method such asguanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry(1979) 18, 5294-5299), a guanidine thiocyanate hot phenol method, aguanidine thiocyanate-guanidine hydrochloride method, a guanidinethiocyanate cesium chloride method, alkali sucrose gradientcentrifugation, or an AGPC method (Chomczynski, P. et al., Anal.Biochem. (1987) 162, 156-159). Thereby, total RNA is prepared. Then, byusing mRNA Purification Kit (manufactured by Pharmacia Corporation) orthe like, target mRNA is prepared. Alternatively, by using QuickPrepmRNA Purification Kit (manufactured by Pharmacia Corporation) or thelike, mRNA may be directly prepared.

By using a reverse transcriptase from thus obtained mRNA, cDNA of anantibody variable region is synthesized. The synthesis of cDNA iscarried out by using AMV Reverse Transcriptase First-strand cDNASynthesis Kit (manufactured by Seikagaku Corporation) or the like.Further, for synthesis and amplification of cDNA, 5&apos; -AmpliFINDERRACE Kit (manufactured by Clontech) and 5&apos; -RACE (Frohman, M. A. etal., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyaysky, A. etal., Nucleic Acids Res. (1989) 17, 2919-2932) utilizing PCR can be used.

From a PCR product obtained as described above, a target DNA fragment ispurified and joined to vector DNA. Further, a recombinant vector isprepared by using the vector DNA. Moreover, a desired recombinant vectoris prepared by introducing thus obtained recombinant vector into E. colior the like and selecting a colony. Furthermore, a target DNA sequenceis checked by a conventionally known method such as a dideoxynucleotidechain termination method. The antibody of the present invention obtainedas described above may be expressed by a conventionally known method andobtained.

In the case where E. coli is used, an antibody gene to be expressed maybe expressed by functionally binding the antibody gene to a downstreamof a common useful promoter. Examples of such a promoter are laczpromoter and araB promoter. In a case where lacz promoter is used, theantibody gene can be expressed by a method of Ward et al. (Nature (1098)341, 544-546; FASEB J. (1992) 6, 2422-2427). Meanwhile, in a case wherearaB promoter is used, the antibody gene can be expressed by a method ofBetter et al. (Science (1988) 240, 1041-1043). Thus produced antibodyaggregates in a cytoplasm and forms an inclusion body. However, thusproduced antibody may be used by appropriately refolding a structure ofthe antibody, after an antibody protein aggregate is isolated.

Note that a signal sequence for antibody secretion may be insertedbetween the promoter and the antigen gene so that the antibody may besecreted into periplasm. This method is conventionally known to a personskilled in the art. As the signal sequence for antibody secretion, pelBsignal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may beused in a case where the antibody is to be produced in a periplasm of E.coli. After the antibody produced in the periplasm is isolated, astructure of the antibody is appropriately refolded and then theantibody is used.

An origin of replication here may be derived from SV40, polyoma virus,adenovirus, bovine papillomavirus (BPV), or the like. Further, foramplifying the number of gene copies in a host cell system, theexpression vector may include, as a selection marker, aminoglycosidetransferase (APH) gene, thimidine kinase (TK) gene, E. colixanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolatereductase (dhfr) gene or the like.

For production of the antibody used in the present invention, anyexpression system such as eucaryotic cells or procaryotic cells may beused. Examples of the eucaryotic cells may be animal cells such asestablished mammalian cell lines, insect cell lines, filamentous fungalcells, and yeast cells. Examples of the procaryotic cells are bacterialcells such as E. coli cells.

Next, thus transformed host cell is cultured in vitro or in vivo, sothat a target antibody is produced. The transformed host cell may becultured by a conventionally known method. For example, as a culturefluid, DMEM, MEM, RPMI1640, or IMDM may be used and at the same time,serum replenisher fluid such as fetal calf serum (FCS) may be used.

Further, the antibody expressed and produced as described above may bepurified so as to be uniform. A method for the purification of thelow-molecular-weight antibody may be a conventionally known method andnot specifically limited. For example, the isolation and purification ofthe low-molecular-weight antibody to be used in the present inventionmay be carried by using an affinity column. As such an affinity column,for example, there are Hyper D, POROS, and Sepharose F.F. (manufacturedby Pharmacia Corporation) as a column employing a protein A column.Other than this, the method may be a general isolation and purificationmethod generally used for proteins and is limited by no means. Forexample, the antibody can be isolated and purified by, other than theaffinity column, one or a combination of two or more selected asappropriate from among a chromatography column, a filter,ultrafiltration, salt precipitation, dialysis and the like (Antibodies ALaboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory,1988).

Further, as described later, in the present invention, thelow-molecular-weight antibody has a carrier binding peptide as a tag.Therefore, by putting solution containing a product from thetransformant in contact with a carrier surface, the low-molecular-weightantibody can be directly adsorbed onto the carrier surface and therebyisolated and purified. The solution containing the product is anysolution which contains a target low-molecular-weight antibody and inwhich an unnecessary impurity from the host is present. Such solutionencompasses, for example, disrupted bacterial cell solution, a solublefraction obtained by centrifugation of disrupted bacterial cellsolution, a material obtained by solubilizing an insoluble fractionobtained by centrifugation of disrupted bacterial cell solution, a cellmembrane fraction, a cell wall fraction, a secretion produced andsecreted from a cell, body fluid, or incompletely purified materialsthereof.

Further, in a case where a large amount of heterogeneous genes areexpressed in a host by introducing recombinant DNA, thelow-molecular-weight antibody may be produced as an insoluble aggregate(inclusion body) for preventing an adverse effect onto the host due toprotein produced. Even in a case where the low-molecular-weight antibodyis produced as an insoluble aggregate, the insoluble aggregate may besolubilized by a denaturing agent and then directly immobilized onto thecarrier surface. Furthermore, in some cases, by removing the denaturingagent from thus solubilized aggregate immobilized onto the carriersurface, the low-molecular-weight antibody may be refolded.

Further, even in a case where a low-molecular-weight antibody has atertiary structure somehow denatured other than the case of theinsoluble aggregate, the low-molecular-weight antibody structure can bedirectly immobilized onto the carrier surface as long as thislow-molecular-weight antibody structure is the low-molecular-weightantibody of the present invention. Further, the low-molecular-weightantibody may be refolded by providing an appropriate refolding buffer tothe low-molecular-weight antibody in an immobilized state. As causes ofthe above denaturization, there are physical causes such as heating,freezing, high pressure, supersonic wave, ultraviolet ray, X-ray,stirring, adsorption, and dilution, and chemical causes such as extremeacidity or alkalinity, organic solvents, heavy metal salts, denaturingagents, and surfactants. Note that the method for obtaining thelow-molecular-weight antibody described above is applicable not only ina case where the low-molecular-weight antibody is obtained but also in acase where an antibody described later, for example, a whole humanantibody, is obtained. In such a case, the above explanation can bereferred to as appropriate.

Further, the antibody immobilized carrier only needs to be provided withat least one antibody immobilized region, and the number of the at leastone antibody immobilized region is not specifically limited. Morepreferably, two or more of the antibody immobilized region are providedin an independent manner.

The wording “two or more . . . are provided in an independent manner”means that a set of the heavy-chain low-molecular-weight antibody andthe light-chain low-molecular-weight antibody are immobilized in oneantibody immobilized region and two or more of such an antibodyimmobilized region are independently present on the carrier. Further,“the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody each is derived from an antibodyrecognizing a different antigen”. This means that each of theheavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody is prepared from an antibody that hasaffinity for a different antigen. For example, there may be an exemplarycase where the heavy-chain low-molecular-weight antibody is derived froman anti-interferon γ (IFNG) antibody and the light-chainlow-molecular-weight antibody is derived from an anti-interleukin 6receptor (IL-6R) antibody. Further, an organism from which the antibodyis originally derived is preferably a human. However, the organism isnot limited to a human but the antibody may be derived from any ofvarious vertebrates such as chickens, mice, rats, rabbits, sheep, andmonkeys.

The carrier in the present invention may be any carrier as long as anantibody can be immobilized to the carrier. Generally, the carrier isinsoluble in water. The carrier may be made of a film, a bead, a gel ora substrate of a material selected from resin, nylon, nitrocellulose,polysaccharide, glass and metal. The carrier is on a support made ofglass, ceramics, metal, plastic, or the like, as needed.

(a) and (b) of FIG. 1 schematically show one example of a structure ofthe antibody-immobilized carrier of the present invention. (a) of FIG. 1is a diagram schematically showing a state where a set of a heavy-chainlow-molecular-weight antibody and a light-chain low-molecular-weightantibody each immobilized onto a carrier reacts with an antigen; and (b)of FIG. 1 is a diagram schematically showing a state in which a numberof combinations of a heavy-chain low-molecular-weight antibody and alight-chain low-molecular-weight antibody are immobilized.

In the antibody-immobilized carrier of the present invention, as shownin (a) and (b) of FIG. 1, each of the heavy-chain low-molecular-weightantibody and the light-chain low-molecular-weight antibody is separatelyimmobilized onto the carrier via a carrier binding peptide. In otherwords, the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody form a pair in one antibody immobilizedregion, and each of the heavy-chain low-molecular-weight antibody andthe light-chain low-molecular-weight antibody is independentlyimmobilized as a separate molecule in the one antibody immobilizedregion. Note that the heavy-chain low-molecular-weight antibody and thelight-chain low-molecular-weight antibody only need to be separatelyimmobilized at immobilization in the antibody immobilized region. Afterthe immobilization in the antibody immobilized region, the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody may be interact with each other or form S—S bond or the like.

Each of the heavy-chain low-molecular-weight antibody and thelight-chain low-molecular-weight antibody is immobilized onto thecarrier via the “carrier binding peptide”. At the immobilization of thelow-molecular-weight antibody, each of the low-molecular-weight antibodyis preferably immobilized in a form in which: C-terminus side is boundto the carrier surface at a right angle with respect to the carriersurface; an antigen binding site is facing outward; and the antibody issterically in a normal position. This is for allowing each of theheavy-chain variable region and the light-chain variable region tocontribute to antigen-antibody reaction. Therefore, preferably, the“carrier binding peptide” is provided to C-terminus side of theheavy-chain variable region in the heavy-chain low-molecular-weightantibody while the “carrier binding peptide” is provided to C-terminusside of the light-chain variable region in the light-chainlow-molecular-weight antibody.

The “carrier binding peptide” here only needs to be a peptide having afunction to bind to a material of the carrier surface. The “carrierbinding peptide” is not specifically limited in other specific aminoacid sequence, length, or the like. Note that in the present invention,“binding” means interacting between the peptide and the carrier surfaceat a strength that is sufficient for use intended in the presentinvention. The term “binding” encompasses a case where the peptide hasaffinity for the carrier surface and adsorbs onto the carrier surface.

For example, preferably, the material of the carrier surface is plasticresin having been hydrophilized by property modification, the plasticresin being polystyrene, polycarbonate, polypropylene, polyethylene,polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA). In a casewhere the above material is used as the material of the carrier surface,the carrier binding peptide is a peptide that has a function to bind toeach of the plastic resin having been hydrophilized. For example, in acase where hydrophilic polystyrene is used as the material of thecarrier surface, a peptide binding to hydrophilic polystyrene isselected.

For the carrier binding peptide, it is possible to use as appropriateany of conventionally known and reported peptides having a function tobind to each plastic resin having been hydrophilized. For example, as apeptide binding to hydrophilic polystyrene (hereinafter, also referredto as “PS-tag”), it is possible to use a peptide described in, forexample, International Application Publication No. WO2009/101807 A1,other than a peptide used in Example described later. Specifically, thepeptide may have a sequence RXXXRRXRR (R: arginine, X: one or acombination of two or more of isoleucine (I), leucine (L), valine (V),alanine (A), glycine (G), methionine (M), serine (S) and threonine (T),shown in SEQ ID NO: 11 in the sequence listing of the presentapplication) in this order from N terminus to C terminus. Morespecifically, it is possible to use a peptide having an amino acidsequence of any of SEQ ID NO: 1 to 20 disclosed in InternationalApplication Publication No. WO2009/101807A1. Note that Example describedbelow employs PS-tag in which X is always isoleucine.

Further, as a peptide having a function to bind to hydrophilicpolycarbonate (PC), it is possible to use, for example, a peptidedisclosed in Publication of Japanese Translation of PCT InternationalApplication, Tokuhyo, No. 2004-518442 (Patent Application No.2003-571248). Further, it is also possible to use a peptide uniquelyfound by the inventors of the present application. This peptide found bythe inventors has an amino acid sequence of any of SEQ ID NO: 12 to 17in the sequence listing of the present application and has a function tobind to PC. Note that the peptide of any of SEQ ID NO: 12 to 17 in thesequence listing of the present application has not been known at filingof the present application.

As a peptide having a function to bind to polymethyl methacrylate (PMMA)is, for example, a peptide described in (Literature of Serizawa et al.(Langmuir 2007, 23, 11127-11133)) or a peptide uniquely found by theinventors of the present invention. This peptide found by the inventorshas an amino acid sequence of SEQ ID NO: 15, 18, or 19 in the sequencelisting of the present application and has a function to bind to PMMA.Note that the peptide of SEQ ID NO: 15, 18, or 19 in the sequencelisting of the present application has not been known at filing of thepresent application.

Among the above examples, it is particularly preferable to use, asdescribed in Example below, (a) as the material of the carrier surface,polystyrene having been hydrophilized and (b) as the carrier bindingpeptide, a peptide binding to hydrophilic polystyrene. This combinationallows formation of a strong bond between the peptide and the carriereven in the present of a surfactant or a denaturing agent.

The carrier binding peptide may directly bind to C terminus of thelow-molecular-weight antibody or may bind to C terminus of thelow-molecular-weight antibody via a suitable linker sequence. Further,to C terminus of the carrier binding peptide, a known tag sequence suchas His tag may be provided.

In general, a surface of plastic resin that is preferable as thematerial of the carrier surface is hydrophobic. However, by carrying outvarious hydrophilization processes onto the surface of plastic resin,the plastic resin can be the carrier having a hydrophilic resin surface.For example, in the case of a polystyrene surface, a carrier having ahydrophilic polystyrene surface can be prepared by performing UV+O₃treatment or plasma oxidation treatment. For a method of thehydrophilization treatment, it is possible to use a method described inInternational Application Publication No. WO2009/101807 A1.

As a carrier (substrate) material of the present invention, it ispossible to use a conventionally known material such as various metalmaterials, a glass plate, and a ceramics plate other than resin. Thecarrier material is not specifically limited. The above-describedplastic resin may be provided on a surface of the carrier material, andthe carrier material with the plastic resin may be used as a substrateof the antibody-immobilized carrier of the present invention. Thecarrier may be in a form of a plate (including a wall surface or abottom surface of a container or a well) or particles. As disclosed inJapanese Patent Application Publication, Tokukai, No. 2007-279018, ifparticulate plastic substrate whose surface has been subjected tohydrophilization is filled in a fluid handling section (each well) of amicrowell plate, the low-molecular-weight antibody can be immobilizedonto the particulate plastic substrate surface only by pouring solutioncontaining the low-molecular-weight antibody into the well.

Further, the antibody-immobilized carrier of the present invention mayalso be used as a sensor chip for a surface plasmon resonance method(SPR method). In this case, for example, a thin film of plastic resinsuch as hydrophilic polystyrene is formed on a gold substrate andantibodies are immobilized onto the thin film.

As described above, in a plurality of antibody immobilized regions onthe antibody-immobilized carrier of the present invention, differentcombinations of the heavy-chain low-molecular-weight antibody and thelight-chain low-molecular-weight antibody each derived from a differentantibody are immobilized. That is, antibody immobilized regions of theantibody-immobilized carrier of the present invention are configuredsuch that antibodies each having different sets of a heavy chain and alight chain are immobilized in the antibody immobilized regions and thenumber of the different sets corresponds to the number of the differentcombinations. The larger the number of the antibody immobilized regionsis, the more efficient screening of antibodies becomes. Therefore, thelarger number of the antibody immobilized regions is preferable.Preferably, the number of the antibody immobilized regions are, forexample, 10², 10⁴, 10⁶, or 10⁷. More specifically, the number ofcombinations of a heavy chain and a light chain of antibodies within aliving human body is considered to be 10⁷. In a case where 10³heavy-chain low-molecular-weight antibodies and 10³ light-chainlow-molecular-weight antibodies are prepared, the number of thecombinations becomes 10⁶. In a case where 10⁴ heavy-chainlow-molecular-weight antibodies and 10³ light-chain low-molecular-weightantibodies are prepared, the number of the combinations becomes 10⁷.Therefore, when the above preferable number of the antibody immobilizedregions are provided, the number of the antibody immobilized regions canbe substantially equal to the number of antibodies within a living humanbody. As a result, on one antibody-immobilized carrier, an antibodyproducing system within a living human body can be reproduced (In VitroDomain Shuffling Technique).

As described in Example below, the inventors of the present inventiondemonstrated that it is possible to create a library of antibodyfragments on a substrate by: (a) recovering antigenic specificity bysuccessively immobilizing and assembling, onto a plastic carrier, Fab Hand Fab L fragments to each of which a hydrophilic polystyrene bindingpeptide is fused; and (b) thoroughly covering all variations of acombination of Fab H and Fab L. In the “In Vitro Domain Shufflingtechnique” developed by the inventors of the present invention, variousheavy-chain low-molecular-weight antibodies and light-chainlow-molecular-weight antibodies are immobilized onto the carrier andthis makes it possible to evaluate antigen binding activities. In otherwords, because an antibody library can be prepared, it becomes possibleto identify an antibody (a combination of a heavy chain and a lightchain) specific to an antigenic protein by carrying out immunoassay byusing thus obtained antibody-immobilized carrier. Therefore, theantibody-immobilized carrier of the present invention is very useful inscreening of antibody drug candidates and screening of antibodies fordiagnosis.

In particular, in a case where a plurality of antibody immobilizedregions are provided on an antibody-immobilized carrier, it ispreferable to manage, by using a known arithmetic and logic unit such asa PC, positional information (coordinate information) of the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody on the carrier. In this configuration, for example, in a casewhere an causal substance (antigen, target molecule) of a specificdisease is put in contact with the antibody-immobilized carrier, it ispossible to specify a heavy chain and a light chain each having highantigenic specificity from coordinates from which a signal is obtained.Further, by putting a link to gene information of the heavy chain andthe light chain that are immobilized, it is possible to obtain anantibody gene having high antigenic specificity.

As described above, according to the present invention, for example, itis possible to significantly reduce time, cost, and work for developingantigen drugs and to thoroughly obtain useful antibodies and antibodygenes. Further, it also becomes possible to develop an antibody medicine(tailor made antibody medicine) corresponding to each individual patientin consideration of individual difference.

<2. Method of Producing Antibody-Immobilized Carrier>

A method, according to the present invention, of producing anantibody-immobilized carrier only need to include the step of:immobilizing a heavy-chain low-molecular-weight antibody including aheavy-chain variable region and a light-chain low-molecular-weightantibody including a light-chain variable region separately onto acarrier, so that an antibody immobilized region is prepared, theheavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody each being derived from an antibodyrecognizing a different antigen. In regard to other steps, conditions,materials, or the like, the method may employ conventionally knownsteps, conditions, materials, or the like, and are not specificallylimited. More preferably, the step of immobilizing is repeated at leasttwo times so that two or more of the antibody immobilized region areprovided in an independent manner. This method of producing anantibody-immobilized carrier can be rephrased as a method of producingthe antibody-immobilized carrier described in the section <1> above.Therefore, the explanation of the section <1> above can be referred toas appropriate for a part overlapping with the explanation of thesection <1> above. Therefore, explanation of the part overlapping isomitted here and explanation in this section is specialized in aproduction method.

For example, in a case where the heavy-chain low-molecular-weightantibody and the light-chain low-molecular-weight antibody are preparedby a genetic recombination technique, the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody are generally obtained as insoluble aggregates as describedabove. In a case where such aggregates are used, the following method(so called a liquid phase refolding method) can be employed. In themethod, first, the aggregates are subjected to solubilization by using adenaturing agent for collecting the aggregates. Then, the denaturingagent is removed by multi-stage dialysis, and then, refolding,purification, and quantitative measurement are carried out.Subsequently, by carrying out the step of immobilizing as describedabove, the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody are immobilized onto the carrier.Alternatively, it is also possible to employ the following method (socalled a solid phase refolding method). In this method, for collectingthe insoluble aggregates, the insoluble aggregates are solubilized byusing a denaturing agent and put in contact with the carrier. Then, thedenaturing agent is removed and refolding is carried out. Either of theliquid-phase refolding method and the solid phase refolding method canbe used in the present invention. However, in view of a yield andefficiency (process and cost), the solid phase refolding method ispreferable.

In other words, the step of immobilizing preferably includes: (a) thefirst sub-step of immobilizing, onto the carrier, insoluble aggregatesof the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody by putting the denatured aggregates incontact with a carrier surface, the denatured insoluble aggregates eachhaving been denatured by a denaturing agent and being in a denaturedstate; and (b) the second sub-step of refolding the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody each in the denatured state, by removing the denaturing agentfrom the heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody that are in the denatured state andimmobilized.

A concentration of the low-molecular-weight antibody contained insolution used in the first sub-step (a) is not specifically limited butcan be set as appropriate. However, the concentration is preferably in arange of 0.1 μg/ml to 500 μg/ml, more preferably in a range of 0.5 μg/mlto 200 μg/ml, and most preferably in a range of 1 μg/ml to 100 μg/ml.

Here, the “denaturing agent” may be a general protein denaturing agent,surfactant, or the like, and is not specifically limited. Examples ofthe “denaturing agent” are protein denaturing agents such as urea andguanidine hydrochloride, and surfactants such as SDS and CHAPS. Aconcentration of the denaturing agent can be set as appropriatedepending on an amount or type of the low-molecular-weight antibodyemployed here, and is not specifically limited. For example, as shown inExample explained later, in a case where the low-molecular-weightantibody is in a range of 5 μg/ml to 100 μg/ml, 0.5 M to 8 M urea ispreferably used as the denaturing agent. More preferably, 0.5 M to 4 Murea is used, and most preferably, 0.5 M to 2 M urea is used. Note thathere, a surfactant such as Tween20 may be used as an aggregationinhibitor. at the same time. In the method of immobilization, forexample, (i) first, a high-concentration denaturing agent (e.g., 8 M)may be used to solubilize the insoluble aggregate; (ii) then thehigh-concentration denaturing agent may be diluted to a preferredconcentration (in a range of 0.5 M to 2 M) of the denaturing agent, and(iii) subsequently, the insoluble aggregate is put in contact with thecarrier for immobilization.

A time for denaturalization or a time for immobilization may be set asappropriate depending on an amount or type of the low-molecular-weightantibody, and is not specifically limited. In the step of immobilizing,a time for putting solution of the low-molecular-weight antibody incontact with the carrier is arranged to be, for example, in a range of10 minutes to 10 hours, more preferably in a range of 30 minutes to 5hours, and most preferably in a range of 1 hour to 3 hours.

A method for removing the denaturing agent in the second sub-step (b)may be a conventionally known method and is not specifically limited.For example, as described in Example below, the denaturing agent can beremoved by general washup with a buffer. Note that in the above steps(a) and (b), a composition of a buffer used in solubilization of thelow-molecular-weight antibody and a composition of washing for removingthe denaturing agnet may be conventionally known compositions,respectively, and are not specifically limited. For example,compositions described in Example below can be suitably used.

Further, preferably, a set of the first sub-step and the second sub-stepin the step of immobilizing is carried out, separately for each of theheavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody. That is, preferably, the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody are immobilized separately in multiple immobilization stages(e.g., two immobilization stages). For example, for the light-chainlow-molecular-weight antibody, the first sub-step (a) is carried out sothat the light-chain low-molecular-weight antibody denatured isimmobilized onto the carrier, and then the second sub-step (b) iscarried out so that the light-chain low-molecular-weight antibodydenatured is refolded. Subsequently, preferably, for the heavy-chainlow-molecular-weight antibody, the first sub-step (a) and the secondsub-step (b) are similarly carried out. This is for the followingreason. That is, as described in Example below, as compared to a casewhere the step of immobilizing is carried out by putting a mixture ofthe heavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody in contact with the carrier, remarkablysuperior antigen binding activity can be obtained in a case where theheavy-chain low-molecular-weight antibody and the light-chainlow-molecular-weight antibody are separately immobilized.

In this way, in a case where the heavy-chain low-molecular-weightantibody and the light-chain low-molecular-weight antibody areimmobilized separately in multiple immobilization stages, the order asto which low-molecular-weight antibody is first immobilized onto thecarrier is not specifically limited. However, preferably, thelight-chain low-molecular-weight antibody is first immobilized bycarrying out the first sub-step and the second sub-step, and then theheavy-chain low-molecular-weight antibody is immobilized by carrying outthe first sub-step and the second sub-step. This is because, asdescribed in Example below, immobilization in this order makes itpossible to further enhance antigen binding ability of antibodies on thecarrier.

According to the method of producing an antibody-immobilized carrier asdescribed above, it is possible to efficiently produce a carrier ontowhich many combinations of a heavy chain and a light chain areimmobilized. Each of the heavy chain and the light chain here is derivedfrom a different antibody and has a lower molecular weight. Note, tomake sure, that the present invention encompasses anantibody-immobilized carrier obtained by the production method describedabove.

<3. Antibody Screening Method>

An antibody screening method of the present invention only needs toinclude the step of: screening a heavy-chain low-molecular-weightantibody and/or a light-chain low-molecular-weight antibody eachrecognizing a specific antigen, by using the antibody-immobilizedcarrier as described above. Other steps, conditions, materials or thelike of the method may be conventionally known steps, conditions,materials or the like, and are not specifically limited. It is possibleto obtain a whole antibody by recloning the heavy-chainlow-molecular-weight antibody and/or the light-chainlow-molecular-weight antibody each selected by the antibody screeningmethod of the present invention.

In a case where the “specific antigen” here is, for example, a targetsubstance of an antibody medicine or a target substance of an antibodyfor diagnosis, it is possible to obtain the antibody medicine or theantibody for diagnosis. For example, in a case where a candidateantibody for an antibody medicine is to be selected, a target substance(antigen) of the antibody medicine is put in contact with theantibody-immobilized carrier as described above and an antibodyrecognizing specifically the antigen should be selected. Note that amethod for evaluating antigen binding activity may be a conventionallyknown method and is not specifically limited. As described in Exampledescribed below, evaluation of the antigen binding activity can becarried out by, for example, ELISA employing a biotinylated antigen.

Further, the “specific antigen” used in the antibody screening method ispreferably labeled for convenience of detection. As a labeling substanceused as a marker is not specifically limited and may be, for example, afluorescent dye, enzyme, protein, radioisotope, a chemiluminescentsubstance, biotin, and a color label substance.

A suitable substance of the fluorescent dye may be a substance that isused for labeling a substance such as a polypeptide or polynucleotidebeing generally an antigen and that is used for detection orquantitative determination. The fluorescent dye is not specificallylimited. Examples of the fluorescent dye are fluorescein isothiocyanate(FITC), EHX (4,7,2′,4′,5′,7′-hexachloro-6-carboxylfluorescein, greenfluorescent dye), fluorescein, NED (product name, manufactured byApplied Biosystems, yellow fluorescent dye) or 6-FAM (product name,manufactured by Applied Biosystems, yellowish green fluorescent dye),rhodamin or a derivative thereof (e.g., tetramethylrhodamin (TMR)),Alexa Fluor (Invitrogen), Cy Dye (GE Healthcare), and Quantum Dot(Invitrogen).

Further, the color label substance may be colloidal metal and coloredlatex. Typical examples of the colloidal metal are platinum colloid andgold colloid. A size of particles of the colloidal metal is generally ina range of approximately 3 nm to 100 nm in diameter. A typical exampleof the colored latex are synthesized latex such as polystyrene latexthat is colored by pigment of each color such as red or green. Thecolored latex may be natural latex such as natural rubber latex. A sizeof the colored latex can be selected from a range of tens of nanometersto hundreds of nanometers in diameter. As these color label substances,commercial products may be directly used. Alternatively, in some cases,the color label substances may be further processed commercial products.As a further alternative, the color label substances themselves may beproduced by a conventionally known method.

The heavy-chain low-molecular-weight antibody and/or the light-chainlow-molecular-weight antibody selected by the antibody screening methodof the present invention or a whole antibody obtained by cloning such alow-molecular-weight antibody can be used for various immunoassaysincluding antigen-antibody reaction, for example, for application toin-vivo treatment and prevention, for application to in-vitro andin-vivo diagnosis, and for application to in-vitro assay and reagent.Note that in a case of the use in application to in-vivo treatment andprevention for human beings and application to diagnosis for humanbeings, it is preferable that the heavy-chain low-molecular-weightantibody and/or the light-chain low-molecular-weight antibody or thewhole antibody is substantially a pure whole human antibody or ahumanized antibody at least 90 to 95% or more, and more preferably 98 to99% or more of which is an antibody portion derived from a human. Aconventionally known method may be used as a technique for (i) analyzingan amino acid sequence of an antibody selected by the antibody screeningmethod of the present invention and (ii) preparing the whole humanantibody or the like.

The present invention encompasses a method of screening a human antibody(whole human antibody) recognizing a specific antigen by using theantibody-immobilized carrier as described above, the screening beingcarried out by using a chimeric antibody or a humanized antibody eachrecognizing the specific antigen.

In view of bioethics, it is impermissible to produce an antibody byimmunizing humans with an antigen of interest. Therefore, all currentlyexisting antibody drugs are developed in the form of chimeric antibodieseach having a variable region of a mouse antibody and a constant regionof a human antibody or humanized antibodies each obtained bytransplanting, into a human antibody, only an antigen binding portion(CDR; complementary determining region) of a mouse antibody. However,there is a strong demand for development of a whole human antibody thathas lower antigenicity and that is safer.

The above screening method of the present invention is developed inresponse to the above demand. The screening method allows obtaining, byusing the In Vitro Domain Shuffling technique employing theantibody-immobilized carrier described above, a whole human antibodyhaving antigenic specificity whose level is substantially equivalent toor higher than that of a chimeric antibody or humanized antibody. Thescreening method more specifically includes the following steps.

That is, in the first aspect, the method of screening a human antibody(whole human antibody) recognizing a specific antigen, the screeningbeing carried out by using a chimeric antibody or a humanized antibodyeach recognizing the specific antigen, the heavy-chainlow-molecular-weight antibody being immobilized onto the at least oneantibody immobilized region and including a heavy-chain variable regionderived from the chimeric antibody or the humanized antibody, thelight-chain low-molecular-weight antibody being a light-chainlow-molecular-weight antibody that is immobilized onto the at least oneantibody immobilized region and that includes a light-chain variableregion derived from a random human antibody, the method includes thesteps of: (i) putting the specific antigen in contact with theantibody-immobilized carrier; (ii) detecting an antibody immobilizedregion recognizing the specific antigen on the antibody-immobilizedcarrier; and (iii) determining a light-chain low-molecular-weightantibody immobilized on the antibody immobilized region detected in thestep (ii), as a candidate for a light-chain variable region of the humanantibody recognizing the specific antigen.

Preferably, the method of screening further includes the steps of: (iv)putting the specific antigen in contact with anotherantibody-immobilized carrier including another antibody immobilizedregion onto which (a) the light-chain low-molecular-weight antibodydetermined as the candidate in the step (iii) and (b) a heavy-chainlow-molecular-weight antibody including a heavy-chain variable regionderived from a random human antibody are immobilized; (v) detecting anantibody immobilized region recognizing the specific antibody on theanother antibody-immobilized carrier; and (vi) determining a heavy-chainlow-molecular-weight antibody immobilized onto the antibody immobilizedregion detected in the step (v), as a candidate for a heavy-chainvariable region of the human antibody recognizing the specific antigen.

The first aspect of the method of screening of the present invention maybe described conceptually as follows. That is, the antibody-immobilizedcarrier in a preferred embodiment includes a plurality of antibodyimmobilized regions in each of which a heavy-chain low-molecular-weightantibody and a light-chain low-molecular-weight antibody are separatelyimmobilized. Here, the heavy-chain low-molecular-weight antibodyimmobilized in the antibody immobilized region is a heavy-chainlow-molecular-weight antibody that includes a heavy-chain variableregion derived from a chimeric antibody or a human antibody. It has beenpreviously confirmed that the chimeric antibody or the human antibodyhere has antigenic specificity against the specific antigen. Meanwhile,the light-chain low-molecular-weight antibody immobilized in a positionfor a light-chain low-molecular-weight antibody is a light-chainlow-molecular-weight antibody including a light-chain variable regionderived from a random human antibody. That is, the antibody-immobilizedcarrier prepared includes plural types of antibody immobilized regionseach including a combination of (a) a known heavy-chainlow-molecular-weight antibody including a heavy-chain variable region(preferably only one type) derived from a chimeric antibody or a humanantibody and (b) a light-chain low-molecular-weight antibody including alight-chain variable region selected discretionarily from a library oflight chains each derived from a human antibody. In regard to a varietyof light chains of human antibodies, it is considered that there areapproximately 10³ light chains. Accordingly, it is preferable to prepareapproximately 10³ antibody immobilized regions. Ultimately, by puttingan antigen in contact with the antibody-immobilized carrier configuredas described above, antibody immobilized regions having high antigenbinding activity are specified. Then, light-chain low-molecular-weightantibodies immobilized in thus specified antibody immobilized regionsare selected. Thus selected light-chain low-molecular-weight antibodiesare determined as candidates for light-chain variable regions of humanantibodies each recognizing the specific antigen.

Subsequently, in antibody immobilized regions of anotherantibody-immobilized carrier additionally prepared, thus selectedlight-chain low-molecular-weight antibodies each including thelight-chain variable region of a human antibody are immobilized.Further, as the heavy-chain low-molecular-weight antibodies, a pluralityof heavy-chain low-molecular-weight antibodies each including aheavy-chain variable region derived from a random human antibody areimmobilized. That is, the antibody-immobilized carrier additionallyprepared includes plural types of antibody immobilized regions eachincluding a combination of (a) a light-chain low-molecular-weightantibody including a light-chain variable region that is derived from ahuman antibody and that has been selected as a candidate and (b) alow-molecular-weight antibody including a heavy-chain variable regionselected discretionarily from a library of heavy chains each derivedfrom a human antibody. In regard to a variety of light chains of humanantibodies, it is considered that there are approximately 10⁴ heavychains. Accordingly, it is preferable to prepare approximately 10⁴antibody immobilized regions. Ultimately, by putting an antigen incontact with the antibody-immobilized carrier configured as describedabove, antibody immobilized regions having high antigen binding activityare specified. Then, heavy-chain low-molecular-weight antibodiesimmobilized in thus specified antibody immobilized regions are selected.Thus selected heavy-chain low-molecular-weight antibodies are determinedas candidates for the heavy-chain variable regions of human antibodieseach recognizing the specific antigen. Note that though it is preferableto prepare approximately 10⁴ antibody immobilized regions, the number ofthe antibody immobilized regions may be arranged to be in a range of twoor more to 10² or in a range of 10² to 10³ for reducing working hoursand labors. Even in such a case, by using the invention of the presentapplication, it is possible to obtain a whole human antibody capable ofproviding a sufficiently excellent effect.

By the above method, a combination of a light chain and a heavy chaineach having high binding activity with respect to a specific antigen canbe selected from a library of human antibodies. By using the combinationof the light chain and the heavy chain selected as described above, awhole antibody having antigen binding activity whose level is equivalentto or higher than that of a chimeric antibody or a humanized antibody.

In the above aspect, first, a candidate for a light chain of a humanantibody having high antigenic specificity is selected by using acombination of a light chain of a human antibody with a heavy chain of achimeric antibody or a humanized antibody. However, the presentinvention is not limited to this aspect. For example, the presentinvention may be configured as follows: first, by using a combination ofa heavy chain of a human antibody with a light chain of a chimericantibody or a humanized antibody, a candidate for a heavy-chainlow-molecular-weight antibody discretionarily selected from a heavychain library of human antibodies is selected; and then, by using acombination of a light chain of a human antibody and thus selectedcandidate for the heavy chain, a candidate for a light chain of a humanantibody having high antigenic specificity may be selected.

That is, in the second aspect, the method of the present invention ofscreening a human antibody recognizing a specific antigen by using theantibody-immobilized carrier as described above, the screening beingcarried out by using a chimeric antibody or a humanized antibody eachrecognizing the specific antigen, the light-chain low-molecular-weightantibody being immobilized onto the at least one antibody immobilizedregion and including a light-chain variable region derived from thechimeric antibody or the humanized antibody, the heavy-chainlow-molecular-weight antibody being a heavy-chain low-molecular-weightantibody that is immobilized onto the at least one antibody immobilizedregion and that includes a heavy-chain variable region derived from arandom human antibody, the method includes the steps of: (i) putting thespecific antigen in contact with the antibody-immobilized carrier; (ii)detecting an antibody immobilized region recognizing the specificantigen on the antibody-immobilized carrier; and (iii) determining aheavy-chain low-molecular-weight antibody immobilized on the antibodyimmobilized region detected in the step (ii), as a candidate for a heavychain variable region of the human antibody recognizing the specificantigen.

Preferably, the method of screening further includes the steps of: (iv)putting the specific antigen in contact with anotherantibody-immobilized carrier including another antibody immobilizedregion onto which (a) the heavy-chain low-molecular-weight antibodydetermined as a candidate in the step (iii) and (b) a light-chainlow-molecular-weight antibody including a light-chain variable regionderived from a random human antibody are immobilized; (v) detecting anantibody immobilized region recognizing the specific antibody on theanother antibody-immobilized carrier; and (vi) determining a light-chainlow-molecular-weight antibody immobilized onto the antibody immobilizedregion detected in the step (v), as a candidate for a light-chainvariable region of the human antibody recognizing the specific antigen.

The above screening method is a two screening stage method arranged suchthat: first, a light chain (or a heavy chain) of a human antibody isonce selected as a candidate; and then, a candidate for a heavy chain(or a light chain) derived from a human antibody having high antigenbinding activity is selected by using a combination of a heavy chain (ora light chain) with the light chain (or a heavy chain) selected as thecandidate. However, the present invention is not limited to this aspect.For example, the method may be configured to include the following steps(three screening stages) of: (a) selecting, as a candidate, a lightchain (or a heavy chain) of a human antibody having high antigen bindingactivity by using a combination of the light chain (or the heavy chain)with a heavy chain (or a light chain) derived from a chimeric antibodyor a humanized antibody; (b) selecting, as a candidate, a heavy chain(or a light chain) of a human antibody having high antigen bindingactivity by using a combination of the heavy chain (or the light chain)with a light chain (or a heavy chain) derived from a chimeric antibodyor a humanized antibody; and (c) obtaining a whole human antibody havingsuperior antigen binding activity by combining the candidates for thelight chain and the heavy chain of the human antibodies, which lightchain and heavy chain of the human antibodies are selected in the steps(a) and (b) that are separately carried out. The above aspect ispreferable in that antigen binding activity can be examined in regard toa plurality of combinations of each of thus selected candidate for thelight chain of the human antibody and thus selected candidate of theheavy chain of the human antibody.

In addition, the present invention encompasses a method of producing ahuman antibody, the method including the step of producing the humanantibody by combining the candidate for the light-chain variable regionof the human antibody and the candidate for the heavy-chain variableregion of the human antibody, the candidate for the light-chain variableregion and the candidate for the heavy-chain variable region beingdetermined by the method of screening as described above. In otherwords, the method of the present invention of producing a human antibodyincludes the method of screening as one step.

A conventionally known method can be employed as a method for producinga whole human antibody from a candidate for a light chain selected froma light chain library of human antibodies and a candidate for a heavychain selected from a heavy chain library of human antibodies. Themethod is not specifically limited. For example, the whole humanantibody can be produced by a cloning technique or a chemical synthesismethod employed in obtaining the “low-molecular-weight antibody”described above. For an explanation of the method of producing the wholehuman antibody, an explanation of the cloning technique or chemicalsynthesis method is referred to. For example, by employing the cloningtechnique, it is possible to collect a peptide having the amino acidsequence of the low-molecular-weight antibody by (i) first, preparingDNA encoding the above antibody, (ii) obtaining recombinant DNA, byinserting thus prepared DNA into an autonomously replicating vector,(iii) introducing as appropriate thus obtained recombinant DNA into ahost such as E. coli, an animal cell or the like and thereby obtaining atransformant, and (iv) culturing the transformant and collecting thepeptide from thus cultured material. Other than the method describedabove, an acellular protein synthesis system or a conventional peptidechemical synthesis method may also be employed. Further, a method forpurifying an antibody may be a conventionally known method and is notspecifically limited.

The present invention is not limited to the description ofconfigurations above, but may be altered by a skilled person within thescope within the description in the present specification. An embodimentbased on a proper combination of technical means disclosed in differentembodiments is encompassed in the technical scope of the presentinvention. All literatures described in the present specification areincorporated herein as references. The following provides descriptionsof the present invention in more detail by providing Example. However,the present invention is not limited to only the following Example.

EXAMPLE 1. Materials

(1) Solubilization Buffer for Solubilizing Inclusion Body (pH 7.5)

6 M guanidine hydrochloride, 10 mM 2-mercaptoethanol, 2×PBS

(2) Binding Buffer For Purifying Fab (pH 7.5) . . . . Solution A 8 Murea, 20 mM imidazole, 2×PBS

(3) Elution Buffer For Purifying Fab (pH 7.5) . . . . Solution B 8 Murea, 400 mM imidazole, 2×PBS

(4) Hydrophilic Polystyrene Support For Solid Phase Refolding (PS Plate)

96-well microplate for tissue culture (AGC Technoglass #3861-096)

(5) Color Solution For ELISA

100 μl of ABTS (Invitrogen: #00-2001) was 100-fold diluted with 0.1 Mcitric acid buffer (pH 4.0) and 0.03% H₂O, and used.

(6) Fab Expression Vector: pET22 (Novagen)

(7) Expression Host: E. coli Rosetta (DE3) (Novagen)

2. Literatures Relevant to Antibody Genes Employed

(1) Anti-RNase Antibody

Katakura Y, Kobayashi E, Kurokawa Y, Omasa T, Fujiyama K, Suga K.

Cloning of cDNA and Characterization of Anti-RNase A Monoclonal Antibody3A21

Journal of Fermentation and Bioengineering. 82, 312-314 (1996)

(2) Anti-CRP Antibody

Dong Hwan Choi, Katakura Y, Ninomiya K, Shioya S. Rational Screening ofAntibodies and Design of Sandwich Enzyme Linked Immunosorbent Assay onthe Basis of a Kinetic Model

Journal of Bioscience and Bioengineering. 105, 261-272 (2008)

(3) Anti-ED-B Antibody and Anti-IFNG Antibody

Alessandro Pini, Francesca Viti, Annalisa Santucci, Barbara Carnemollai,Luciano Zardii, Paolo Neri, and Dario Neri

Design and Use of a Phage Display Library

The Journal of Biological Chemistry. 273, 21769-21776 (1998)

3. Preparation Of Low-Molecular-Weight Antibody (Fab and PS-tag FusedFab)

All of the above literatures are relevant to scFv genes. Accordingly, inthe present example, Fab gene was prepared by (i) isolating genescorresponding to VH and VL regions from scFv gene by PCR and (ii) fusingthus isolated genes with genes of CH₁ region and C_(k) region,respectively. The followings are amino acid sequences of Fab and PS-tagfused Fab employed in Example. Note that N terminus of each of the Faband PS-tag fused Fab employed here is arranged to be Methionine (M) thatis a start codon. Note further that in a case without PS-tag, histidinetag (H×6) is added to an end of a sequence derived from pET22 at Cterminus. Meanwhile, in a case with PS-tag, PS-tag sequence is addedbetween sequences derived from pET and histidine tag (H×6) is added to Cterminus.

Fab-H derived from mouse anti-RNase antibody (Fab H) . . . SEQ ID NO: 1

Fab-H derived from PS-tag fused mouse anti-RNase antibody (Fab H-PS) . .. SEQ ID NO: 2

Fab-L derived from mouse anti-RNase antibody (Fab L) . . . SEQ ID NO: 3

Fab-L derived from PS-tag fused mouse anti-RNase (Fab L-PS) . . . SEQ IDNO: 4

Fab-H derived from PS-tag fused mouse anti-CRP antibody (Fab H-PS) . . .SEQ ID NO: 5

Fab-L derived from PS-tag fused mouse anti-CRP antibody (Fab L-PS) . . .SEQ ID NO: 6

Fab-H derived from PS-tag fused human anti-ED-B antibody (Fab H-PS) . .. SEQ ID NO: 7

Fab-L derived from PS-tag fused human anti-ED-B antibody (Fab L-PS) . .. SEQ ID NO: 8

Fab-H derived from PS-tag fused human anti-IFNG antibody (Fab H-PS) . .. SEQ ID NO: 9

Fab-L derived from PS-tag fused human anti-IFNG antibody (Fab L-PS) . .. SEQ ID NO: 10

The following describes a method for preparing a specificlow-molecular-weight antibody (Fab and PS-tag fused Fab).

(A) Culture of E. coli

First, recombinant E. coli was inoculated onto 10 ml of 2×YT medium(containing Amp and Cm) and precultured at 37° C. overnight. Then, thusobtained precultured solution was added to 50 ml of Overnight Expressmedium (Novagen) (containing Amp and Cm) so that OD₆₀₀ became equal to0.1. Then, 24-hour culture at 37° C. at 200 rpm was carried out. Afterthis culture, cultured solution was transferred to a centrifugation tubeand 20-minute centrifugation was carried out. Then, supernatant wasremoved. Note that antibiotic substances Amp and Cm were added so that aconcentration of Amp became 50 μg/ml and a concentration of Cm became 34μg/ml in the 2×YT medium.

(B) Collection Of Inclusion Body

First, to the E. coli cells in the form of a pellet, 2.5 ml of BugBuster(Novagen), 1 mg/ml of lysozym, and 2.0 μl of Benzonase Nuclease(Novagen) were added and thus obtained mixture was vortexed. Then, aresulting mixture was poured separately into Eppendorf tubes andsubjected to centrifugation (at 4° C., at 20000×g, and for 20 minutes).Further, after a supernatant was removed and 800 μl of distilled waterwas added, a resulting mixture was vortexed and subjected again tocentrifugation (at 4° C., at 20000×g, and for 20 minutes). Subsequently,after a supernatant was removed, an inclusion body was collected.Ultimately, 5 ml of solubilization buffer was added, so that theinclusion body was dissolved.

(C) Purification By Affinity Chromatography

The following procedure was used for purifying low-molecular-weightantibodies that had been solubilized and that was in a denatured state.First, solutions A and B were prepared. Then, a chromatography system(AKTA) was turned on and the solutions A and B were set in Lines A andB, respectively. Contents of Lines A and B were replaced with solutionsA and B, respectively. Then, His Trap™ HP column (GE HealthCare) wasattached. Further, the solution A was supplied into the His Trap™ HPcolumn at a flow rate of 1 ml/min so that an inside of this column wasequilibrated. Furthermore, the low-molecular-weight antibodies havingbeen solubilized was supplied at a flow rate of 1 ml/min and adsorbed tothe column. Then, the solution A was supplied to the column at a flowrate of 1 ml/min and the column was washed. Subsequently, the solution Bwas supplied into the column at a flow rate of 1 ml/min and the columnwas washed. Then, Fab and PS-tag fused Fab were collected. After thecollection, the low-molecular-weight antibodies were subjected toovernight dialysis against 8 M urea-1×PBS.

4. Preparation of Biotinylated Antigen

First, antigens (1 mg/ml) was dialyzed against 1 L of 1×PBS. Then, 1 mgof biotinamidocaproate N-hydroxysuccinimide ester was measured andtaken. To this biotinamidocaproate N-hydroxysuccinimide ester, 10 μl ofN,N-dimethylformamide was added and the biotinamidocaproateN-hydroxysuccinimide ester was dissolved (solution C). The antigenshaving been dialyzed as described above were transferred into a samplebottle. To this bottle, 10 μl of the solution C was added and gentlystirred at a room temperature for one hour. Subsequently, overnightdialysis against 1 L of 1×PBS was carried out.

5. Antibody-Immobilized Carrier Employing Low-Molecular-Weight AntibodyDerived from Mouse Anti-RNase Antibody

A low-molecular-weight antibody derived from a mouse anti-RNase antibodywas immobilized onto a hydrophilic PS plate by a solid phase refoldingmethod. More specifically, first, the low-molecular-weight antibody FabH or Fab H-PS (or Fab L or Fab L-PS) was diluted so that: (a) a finalconcentration of the low-molecular-weight antibody Fab H or Fab H-PS (orFab L or Fab L-PS) became 100 μg/ml; (b) a final concentration of ureabecame 4 M; and (c) a final concentration of Tween 20 became 1%. Then,thus obtained mixture was incubated at a room temperature for 10minutes. Then, 100 μl of the mixture was put on the hydrophilic PSplate, and incubated at 25° C. for one hour. Then, the hydrophilic PSplate was washed five times with 0.1% PEST, and the low-molecular-weightantibody Fab H was refolded on the hydrophilic PS plate. This operationwas repeatedly carried out for each of Fab L, Fab L-PS and Fab H-PS andeach of Fab L, Fab L-PS and Fab H-PS was immobilized onto a hydrophilicPS plate.

The following 6 ways of antibody-immobilized carriers were prepared:

Fab H-immobilized carrier (H)

Fab L-immobilized carrier (L)

Fab H-PS-immobilized carrier (H-PS)

Fab L-PS-immobilized carrier (L-PS)

Carrier (H-PS/L-PS) onto which Fab L-PS was immobilized after Fab H-PShad been immobilized onto the carrier

Carrier (L-PS/H-PS) onto which Fab H-PS was immobilized after Fab L-PShad been immobilized onto the carrier

By using these antibody-immobilized carriers, each antigen bindingactivity of each low-molecular-weight antibody was examined by ELISA.More specifically, first, 300 μl of 2% BSA-PBST was added to the PSplate onto which the low-molecular-weight antibody was immobilized.Then, one-hour incubation at 25° C. was carried out, and the PS platewas washed with PBST five times. Further, 100 μl of biotinylated antigen(mouse RNase) diluted with 0.2% BSA-PBST to a concentration in a rangeof 0 μg/ml to 5 μg/ml, and one-hour incubation at 25° C. was carriedout. Furthermore, the PS plate was washed with PBST five times. Next,100 μl of HRP labeled streptavidin 5000-fold diluted with 0.2% BSA-PBSTwas added, and one-hour incubation at 25° C. was carried out.Subsequently, the PS plate was washed with PBST five times. Further, 100μl of color solution was added, and 30-minute incubation at 25° C. wascarried out. Then, absorbancy was measured. FIG. 2 show a result of thismeasurement. As shown in FIG. 2, antigen binding activity was obtainedonly in L-PS/H-PS.

6. Examination on Conditions for Solid Phase Refolding

Regarding conditions for solid phase refolding at immobilization of alow-molecular-weight antibody derived from mouse anti-RNase antibodyonto a hydrophilic PS plate, examination was carried out. The followingis a specific method of the examination. First, the low-molecular-weightantibody was immobilized onto the hydrophilic PS plate in the samemanner as described in the section <5> above except that a finalconcentration of the low-molecular-weight antibody (Fab H, Fab H-PS, FabL or Fab L-PS) was set at 5 μg/ml and a final concentration of urea wasset at 4M, 2M, 1M, or 0.5M. For each of different combinations oflow-molecular-weight antibodies, the operation was repeatedly carriedout. As a result, the following 5 types of antibody-immobilized carrierswere prepared.

Carrier (H+L-PS) onto which Fab L-PS was immobilized after Fab H hadbeen immobiliazed onto the carrier

Carrier (L-PS+H) onto which Fab H was immobilized after Fab L-PS hadbeen immobilized onto the carrier

Carrier (L+H-PS) onto which Fab H-PS was immobilized after Fab L hadbeen immobilized onto the carrier

Carrier (H-PS+L-PS) onto which Fab L-PS was immobilized after Fab H-PShad been immobilized onto the carrier

Carrier (L-PS+H-PS) onto which Fab H-PS was immobilized after Fab L-PShad been immobilized onto the carrier

By using these antibody-immobilized carriers, antigen binding activityof each low-molecular-weight antibody was examined by ELISA. Here, ELISAwas carried out in the same manner as described in the section <5>except that the concentration of the biotinylated antigen (mouse RNase)employed was set at 1 μg/ml. FIG. 3 shows a result of this examination.

As shown in FIG. 3, in a case where the concentration of urea was in arange of 0.5 M to 2 M, antigen binding activity was higher in the orderof L-PS+H-PS, H-PS+L-PS, L+H-PS, H+L-PS, and L-PS+H. Note that in a casewhere the concentration of urea was 4 M, antigen binding activity wasseen only in L-PS+H-PS.

7. Antibody-Immobilized Carriers Each Employing Low-Molecular-WeightAntibody Derived from Mouse Anti-CRP Antibody, Mouse Anti-RNaseAntibody, Human Anti-IFNG Antibody Or Human Anti-ED-B Antibody

Antibody-immobilized carriers were prepared by immobilizing one or acombination of Fab H-PS and Fab L-PS each derived from any of mouseanti-CRP antibody, mouse anti-RNase antibody, human anti-IFNG antibodyand human anti-ED-B antibody. First, after one of the above types of FabL-PS was immobilized onto a hydrophilic PS plate, one of the above typesof Fab H-PS was immobilized. This immobilization was carried out in thesame manner as described in the above sections <5> and <6> except thatthe final concentration of each of Fab L-PS and Fab H-PS was set at 5μg/ml and the final concentration of urea was set at 2 M. The aboveoperation was repeatedly carried out for each of different combinationsof one of the above types of Fab L-PS and one of the above types of FabH-PS. As a result, 16 types of the antibody-immobilized carrierscovering all combinations of Fab L-PS and Fab H-PS of the above typeswere prepared. In addition, 8 types of antibody-immobilized carriers oneach of which only one of Fab L-PS and Fab H-PS of the above types wasimmobilized.

Antigen binding activity of each of Fab L-PS and Fab H-PS above wasexamined by ELISA by using mouse RNase, mouse CRP, human ED-B and humanIFNG as antigens. Here, ELISA was carried out in the same manner asdescribed in the section <6>. FIGS. 4 to 7 show detection results. InFIGS. 4 to 7, “w/o H” means that Fab H-PS was not immobilized and onlyFab L-PS was immobilized. Similarly, “w/o L” indicates that Fab L-PS wasnot immobilized and only Fab H-PS was immobilized. Moreover, in a casewhere a vertical axis of the graph is labeled “H ED-B”, a bar of “LED-B” shows a result of antigen binding activity of a PS plate ontowhich Fab L-PS and Fab H-PS each derived from human anti-ED-B antibodywere immobilized.

As shown in FIG. 4, in a case where mouse RNase was used as an antigen,only the PS plate onto which Fab L-PS and Fab H-PS each derived frommouse anti-RNase antibody were immobilized had antigen binding activity.As shown in FIG. 5, in a case where mouse CRP was used as an antigen, inregard to light chains, Fab L-PS derived from mouse anti-CRP antibodyhad high antigen binding activity. However, in this case, in regard toheavy chains, Fab H-PS derived from human anti-ED-B antibody had highantigen binding activity. A combination of Fab L-PS derived from mouseanti-CRP antibody and Fab H-PS derived from human anti-ED-B antibody hadthe highest antigen binding activity. FIG. 6 shows a case where humanED-B was used as an antigen. In this case, Fab L-PS derived from mouseanti-CRP antibody had high antigen binding activity in a case where theheavy chain was Fab H-PS that was derived from an antibody except humananti-ED-B antibody. Meanwhile, in regard to a combination of a heavychain and a light cahin, a combination of Fab L-PS and Fab H-PS eachderived from human anti-ED-B antibody had the highest antigen bindingactivity. FIG. 7 shows a case where human IFNG was used as an antigen,specificity was found in neither Fab H-PS nor Fab L-PS and, in thiscase, it was considered that binding to the antigen occurs depending onFab H.

8. Examination of Antigen Binding Activity of Low-Molecular-WeightAntibody in Relation to Difference in Immobilization Method

Examination was carried out on difference in antigen binding activitydue to difference in immobilization method in preparing a carrier ontowhich one or a combination of Fab H, Fab L, Fab H-PS, and Fab L-PS eachderived from mouse anti-RNase antibody was immobilized. Specifically,the following cases (i) to (iii) were compared: (i) a case where aheavy-chain low-molecular-weight antibody or a light-chainlow-molecular-weight antibody was solely immobilized; (ii) a case wherea heavy-chain low-molecular-weight antibody and a light-chainlow-molecular-weight antibody in a mixed state were immobilized at thesame time; and (iii) a case where a heavy-chain low-molecular-weightantibody and a light-chain low-molecular-weight antibody weresuccessively immobilized in multiple stages. A specific method for theexamination was as follows:

(i) Case where Low-Molecular-Weight Antibody Alone was Immobilized

First, any one of Fab H, Fab H-PS, Fab L and Fab L-PS was diluted sothat: (a) a final concentration of this low-molecular-weight antibodywas 200 μg/ml; (b) a final concentration of urea was 4 M; and (c) afinal concentration of Tween 20 was 1%. Then, 10-minute incubation at aroom temperature was carried out. Further, 100 μl of thus obtainedmixture was provided on a hydrophilic PS plate and one-hour incubationat 25° C. was carried out. Then, the hydrophilic PS plate was washedwith PBST five times. Further, the any one of Fab H, Fab H-PS, Fab L andFab L-PS was refolded on the hydrophilic PS plate. (a) of FIG. 8 shows aresult of the examination on antigen binding activity.

(ii) Case where Low-Molecular-Weight Antibodies Mixed were Immobilized

First, a combination of (a) Fab H or Fab H-PS and (b) Fab L or Fab L-PSwas diluted so that: (a) a final concentration of each of theselow-molecular-weight antibodies was 200 μg/ml (a final concentration ofa sum of the combination of these low-molecular-weight antibodies was400 μg/ml); (b) a final concentration of urea was 4 M; and (c) a finalconcentration of Tween 20 was 1%. Then, 10-minutes incubation at a roomtemperature was carried out. Further, 100 μl of thus obtained mixturewas provided on a hydrophilic PS plate and one-hour incubation at 25° C.was carried out. Then, the hydrophilic PS plate was washed with PBSTfive times. Further, the low-molecular-weight antibodies of thecombination were refolded on the hydrophilic PS plate. (b) of FIG. 8shows a result of the examination on antigen binding activity.

(iii) Case where Low-Molecular-Weight Antibodies were SuccessivelyImmobilized in Multiple Stages

Except that the final concentration of each of the low-molecular-weightantibodies was arranged to be 200 μg/ml, the low-molecular-weightantibodies were immobilized in the same manner as described in thesection <5> above. (c) of FIG. 8 shows a result of antigen bindingactivity.

As shown in (a) to (c) of FIG. 8, in both of (i) the case where alow-molecular-weight antibody alone was immobilized and (ii) the casewhere low-molecular-weight antibodies mixed were immobilized, antigenbinding activity was hardly found. Only in (iii) the case where thelow-molecular-weight antibodies were successively immobilized inmultiple stages, antigen binding activity was found. Particularly in acase where Fab H or Fab H-PS was immobilized onto a carrier after Fab Lor Fab L-PS had been immobilized onto the carrier, excellent antigenbinding activity was found.

9. Experiment of Screening of Anti-AFP Antibody from Mouse AntibodyLibrary

First, first-stage screening was carried out. The first-stage screeningwas screening of a heavy chain (H chain) and a light chain (L chain)each having affinity for an antigen. The following explains specificprocedures.

(1) Amplification of Antibody Gene from Mouse Immunized with AFP;

As an antigen, α-fetoprotein (AFP) that is a diagnosis marker for livercancer was used. Every week, a mouse was immunized with 50 μg of AFP,and the mouse was immunized four times in total. Then, in the fifthweek, a spleen was taken out. Then, Total RNA in the spleen wascollected and gene clusters of Fab H and Fab L of an antibody wasamplified by RT-PCR. The gene clusters of Fab H and Fab L were subjectedto ligation at Nde I/Not I site of PS-tag fused protein expressionvector pET-PS19-6. Then, E. coli BL21 (DE3) Rosetta was transformed byusing the vector having been subjected to the ligation. Further, asingle colony of thus transformed E. coli was formed on an LB-ampicillinplate. Then, E. coli into which a Fab H gene cluster was introduced wasdefined as a Fab H-PS E. coli library (1×10⁵ colonies). Meanwhile, E.coli into which a Fab L gene cluster was introduced was defined as a FabL-PS E. coli library (1×10⁵ colonies).

(2) High-Throughput Production of PS-Tag Fused Fab H (Fab H-PS) andPS-Tag Fused Fab L (Fab L-PS) by using Microplate;

First, into each well of a 96-well deep well plate (manufactured byGreiner Bio-One, 780271), 1 ml of Overnight Express TB medium (Merk) wasprovided. The Overnight Express TB medium contained 50 μg/ml ofampicillin and 34 μg/ml of chloramphenicol. The number of such a 96-welldeep well plate prepared was 20. Then, a single colony was picked fromthe Fab H-PS E. coli library obtained by forming single colonies on anLB-ampicillin plate. Further, thus picked single colony was inoculatedin each well of the above-described deep well plate. In a similarmanner, a single colony from the Fab L-PS E. coli library was alsoinoculated. More specifically, for each of the Fab H-PS E. coli libraryand the Fab L-PS E. coli library, 10 plates (960 colonies) were used forinoculation. Then, the 96-well deep well plate was incubated at 37° C.at 1400 rpm for 24 hours.

(3) Bacterial Cell Disruption and Solubilization Of Inclusion Body;

Next, the 96-well deep well plate was subjected to centrifugation at5000 rpm for 20 min, and supernatant was removed. Then, 200 μl ofdisrupted cell solution (20 ml of BugBuster, 20 mg of Lysozyme, and 6 μlof benzonaze) was added to each well, and the deep well plate was shakenat 37° C. at 1800 rpm for one hour. Furthermore, after centrifugation at5000 rpm for 20 min, supernatant was removed. Then, 200 μl of ionexchanged water was added to each well and suspended. The aboveoperation was repeated twice. Subsequently, after centrifugation at 5000rpm for 20 min was carried out, 500 μl of a solubilizing solution (50 mlof 8 M Urea PBS, and 35 μl of mercaptoethanol) was added to each well.Then, the deep well plate was shaken at 25° C. at 14000 rpm for onehour. Subsequently, centrifugation at 5000 rpm for 20 min was carriedout, and Fab H-PS and Fab L-PS contained in supernatant was purified asfollows.

(4) Purification of Fab H-PS and Fab L-PS by Using Filter Plate;

To a 96-well filter plate (Whatman), 200 μl/well of 50% Ni Sepharose 4B(GE HealthCare) resin was added. Then, equilibration was performed byusing 200 μl of Binding Buffer 1 (8 M urea-2×PBS, 20 μM imidazole). Intoeach well of the 96-well filter plate, 500 μl of a sample from the96-well deep well plate was added. By pipetting, the above resin and thesample were gently mixed and then 20-minute incubation was carried out.Then, sample solution was sucked by an aspirator and removed. Next, thefilter plate was washed once with 400 μl of Binding Buffer 1, andfurther washed twice with 200 μl of Binding Buffer 1. In addition, thefilter plate was washed with Binding Buffer 2 (8 M urea-1×PBS, and 20 μMof imidazole), and subsequently, solution was removed by an aspirator.

Then, (i) to each well of the filter plate, 250 μl of Elution Buffer (8M urea-400 mM of imidazole-1×PBS) was added and incubation was carriedout for 20 minutes so that Fab H-PS and Fab L-PS each adsorbed to resinwas eluted. Further, (ii) the 96-well filter plate was overlapped with a800-μl 96-well deep well plate (Greiner Bio-One) and thus elutedsolution was collected by centrifugation (500 g, for 5 min) into the800-μl 96-well deep well plate for collection. This operation wasrepeated twice. Subsequently, the operations (i) and (ii) above wererepeated once again.

(5) Isolation of Clone with High Affinity for AFP from Fab H-PS Libraryand Fab L-PS Library

As a 96-well PS plate for screening, a Black plate (BD Falcon #353241)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 75 μl of 1.33% Tween PBS wasadded to each well, 25 μl of a Fab H-PS library or a mouse H chainlibrary was added to each well. Then, the Fab H-PS library or the mouseH chain library was incubated at 4° C. overnight. Then, after the PSplate was washed with 0.1% Tween PBS, 270 μl of 2% BSA-0.1% Tween PBSwas added to each well. Next, the PS plate was subjected to blocking forone hour. Further, after the PS plate was washed with 0.1% Tween PBS, 1μg/ml Alexa Fluor647-labeled AFP was prepared by using 0.2% BSA-0.1%Tween PBS. After 100 μl of thus obtained Alexa Fluor647-labeled AFP wasadded to each well, one-hour incubation was carried out while light wasshielded. Subsequently, after the PS plate was washed with 0.1% TweenPBS, fluorescence intensity was measured by using a fluorescence platereader (TECAN infinite M200) (excitation wavelength: 645 nm,fluorescence wavelength: 678 nm).

Top 40 types of Fab H-PS clones and top 30 types of Fab L-PS clones inthe order of higher fluorescence intensity were selected and used in thefollowing steps. FIGS. 9 and 10 show results of the following steps.Note that bar graphs of FIGS. 9 and 10 show adsorbed amount (scale onthe left side) and dots shows fluorescence intensity (scale on the rightside). Note that an average signal intensity of 960 clones of H chainwas 1086. Meanwhile, an average signal intensity (fluorescenceintensity) of 960 clones of L chain was 1028.

(6) Production of Fab Library Plate by Using In Vitro Domain Shufflingand Screening of Fab with High Affinity for AFP;

By using the 40 types of Fab H-PS clones and the 30 types of Fab L-PSclones selected above, 1200 types (H chain: 40 types×L chain: 30 types)of antibody libraries were prepared on a substrate. Then, antigenbinding ability was evaluated (second-stage screening). The followingexplains specific procedure for the evaluation.

As a 384-well PS plate for screening, a Black plate (BD Falcon #353285)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 30 μl of 1.33% Tween PBS wasadded to each well, 10 μl of each of the top 30 types of Fab L-PS clonesfrom the Fab L-PS library was added to each well. Then, the 384-well PSplate was incubated at 25° C. for 2 hours. Further, after 30 μl of 1.33%Tween PBS was added to each well, 10 μl of each of the top 40 types ofclones from the Fab H-PS library was added to each well. Then, 2-hourincubation at 25° C. was carried out. Next, after the PS plate waswashed with 0.1% Tween PBS, 80 μl of 2% BSA-0.1% Tween PBS was added toeach well. Next, this PS plate was subjected to blocking for one hour.Further, after the PS plate was washed with 0.1% Tween PBS, 1 μg/mlAlexa Fluor647-labeled AFP was prepared by using 0.2% BSA-0.1% TweenPBS. After 100 μl of thus obtained Alexa Fluor647-labeled AFP was addedto each well, one-hour incubation was carried out while light wasshielded. Subsequently, after the PS plate was washed with 0.1% TweenPBS, fluorescence intensity was measured by using a fluorescence platereader (TECAN infinite M200) (excitation wavelength: 645 nm,fluorescence wavelength: 678 nm).

Consequently, a heavy-chain and light-chain combination whose signal offluorescence intensity was 5000 or higher was determined as an anti-AFPantibody. As a result, the following 9 combinations were determined asanti-AFP specific antibodies for liver cancer: (H3, L4), (H3, L6), (H3,L12), (H6, L6), (H8, L6), (H8, L15), (H19, L9), (H20, L9) and (H35, L6).Note that H in parentheses means a heavy chain and numerals inparentheses show the order of H chains shown in FIG. 9. Similarly, L inparentheses means a light chain and numerals in parentheses show theorder of L chain shown in FIG. 10. In other words, each of theseheavy-chain and light-chain combinations was found to be useful as ananti-AFP antibody.

10. Conversion of Chimeric Antibody into Human Antibody by Using HumanAntibody Library

By using In Vitro Domain Shuffling, the following experiment was carriedout for obtaining a whole human antibody having antigenic specificitywhose level is equivalent to or higher than that of a chimeric antibody.

(1) Amplification of Human Antibody Gene;

From human spleen Total RNA (Clontech, #636525), gene clusters of Fab Hand Fab L of a human antibody was amplified by RT-PCR. The gene clustersof Fab H and Fab L were subjected to ligation at Nde I/Not I site ofPS-tag fused protein expression vector pET-PS 19-6. Then, E. coli BL21(DE3) Rosetta was transformed by using the vector having been subjectedto the ligation. Further, a single colony of thus transformed E. coliwas formed on an LB-ampicillin plate. Then, E. coli into which a Fab Hgene cluster was introduced was defined as a Fab H-PS E. coli library(1×10⁶ colonies). Meanwhile, E. coli into which a Fab L gene cluster wasintroduced was defined as a Fab L-PS E. coli library (1×10⁶ colonies).

(2) High-Throughput Production of PS-Tag Fused Fab H (Fab H-PS) andPS-Tag Fused Fab L (Fab L-PS) by Using Microplate;

First, into each well of a 96-well deep well plate (manufactured byGreiner Bio-One, 780271), 1 ml of Overnight Express TB medium (Merk) wasprovided. The Overnight Express TB medium contained 50 μg/ml ofampicillin and 34 μg/ml of chloramphenicol. The number of such a 96-welldeep well plate prepared was 20. Then, a single colony was picked fromthe Fab H-PS E. coli library obtained by forming single colonies on anLB-ampicillin plate. Further, thus picked single colony was inoculatedin each well of the above-described deep well plate. In a similarmanner, a single colony from the Fab L-PS E. coli library was alsoinoculated. (10 plates (960 colonies) for each of the Fab H-PS E. colilibrary and the Fab L-PS E. coli library). Then, the 96-well deep wellplate was incubated at 37° C. at 1400 rpm for 24 hours.

(3) Bacterial Cell Disruption and Solubilization of Inclusion Body;

Next, the 96-well deep well plate was subjected to centrifugation at5000 rpm for 20 min, and supernatant was removed. Then, 200 μl ofdisrupted cell solution (20 ml of Bugbuster, 20 mg of Lysozyme, and 6 μlof benzonaze) was added to each well, and the deep well plate was shakenat 37° C. at 1800 rpm for one hour. Furthermore, after centrifugation at5000 rpm for 20 min, supernatant was removed. Then, 200 μl of ionexchanged water was added to each well and suspended. The aboveoperation was repeated twice. Subsequently, after centrifugation at 5000rpm for 20 min was carried out, 500 μl of a solubilizing solution (50 mlof 8 M Urea PBS, and 35 μl of mercaptoethanol) was added to each well.Then, the deep well plate was shaken at 25° C. at 14000 rpm for onehour. Subsequently, centrifugation at 5000 rpm for 20 min was carriedout, and Fab H-PS and Fab L-PS contained in supernatant was purified asfollows.

(4) Purification of Fab H-PS and Fab L-PS by Using Filter Plate;

To a 96-well filter plate (Whatman), 200 μl/well of 50% Ni Sepharose 4B(GE HealthCare) resin was added. Then, equilibration was performed byusing 200 μl of Binding Buffer 1 (8 M urea-2×PBS, 20 μM imidazole). Intoeach well of the 96-well filter plate, 500 μl of a sample from the96-well deep well plate was added. By pipetting, the above resin and thesample were gently mixed and then 20-minute incubation was carried out.Then, sample solution was sucked by an aspirator and removed. Next, thefilter plate was washed once with 400 μl of Binding Buffer 1, andfurther washed twice with 200 μl of Binding Buffer 1. In addition, thefilter plate was washed with Binding Buffer 2 (8 M urea-1×PBS, and 20 μMof imidazole), and subsequently, solution was removed by an aspirator.

Then, (i) to each well of the filter plate, 250 μl of Elution Buffer (8M urea-400 mM of imidazole-1×PBS) was added and incubation was carriedout for 20 minutes so that Fab H-PS and Fab L-PS each adsorbed to resinwas eluted. Further, (ii) the 96-well filter plate was overlapped with a800-μl 96-well deep well plate (Greiner Bio-One) and thus elutedsolution was collected by centrifugation (500 g, for 5 min) into the800-μl 96-well deep well plate for collection. This operation wasrepeated twice. Subsequently, the operations (i) and (ii) above wererepeated once again.

(5) Evaluation of Rituxan (Registered Trademark) Chimeric Fab H-PS/HumanFab L-PS Library by Using In Vitro Domain Shuffling (Selection of 95Clones from Among 960 Clones);

By using, as a model antibody, Rituxan (registered trademark) that hasbeen available on the market, an attempt was made to obtain a wholehuman antibody having antigenic specificity whose level was equivalentto or higher than that of a chimeric antibody. Note that Rituxan(registered trademark) is an antibody medicine that targets CD20 andthat is applied to cell lymphoma. Further, note that an epitope site ofCD20 antigen has been already disclosed. Therefore, a fluorescencelabeled epitope peptide was prepared by (i) synthesizing only an epitopepeptide by solid-phase synthesis and (ii) adding FITC to N terminus ofthe epitope peptide. Then, this fluorescence labeled epitope peptide wasused as an antigen. The following describes a specific procedure.

As a 384-well PS plate for screening, a Black plate (BD Falcon #353285)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 27 μl of 1.33% Tween PBS wasadded to each well, 9 μl of the human Fab L-PS library was added to eachwell. Then, 4 μl of Rituxan (registered trademark) chimeric Fab H-PS wasadded to each well. Rituxan (registered trademark) chimeric Fab H-PShere had been prepared so that a concentration of the Rituxan(registered trademark) chimeric Fab H-PS was 250 μg/ml in 2 M urea and1% Tween PBS. Then, two-hour incubation was carried out. Next, after thePS plate was washed with 0.1% Tween PBS, 80 μl of 2% BSA-0.1% Tween PBSwas added to each well. Then, the PS plate was subjected to blocking forone hour. Further, after the PS plate was washed with 0.1% Tween PBS, 1μg/ml FITC labeled antigen solution was prepared. This FITC labeledantigen solution contained 0.1% Tween 20 and 5% human serum. Then, 40 μlof thus prepared FITC labeled antigen solution was added to each well.Then, while light was shielded, one-hour incubation was carried out.Subsequently, after the PS plate was washed with 0.1% Tween PBS,fluorescence intensity was measured by using a fluorescence plate reader(TECAN infinite M200) (excitation wavelength: 486 nm, fluorescencewavelength: 520 nm).

FIG. 11 shows a result of combining a chimeric H chain and a human Lchain library (960 types). In FIG. 11, a vertical axis shows fluorescentintensity, while a horizontal axis shows types of human L chains. Inregard to combinations of the chimeric H chain and the human L chainlibrary, the highest fluorescence intensity was 6435 while an averagefluorescence intensity was 2466. From the human L chains whosefluorescence intensity was 4000 or higher among the 960 clones incombination with the chimeric H chain, top 95 clones in the order offluorescence intensity detected were selected.

(6) Evaluation of Rituxan (Registered Trademark) Chimeric Fab H-PS/Top95 Clones of Human Fab L-PS Library by Using In Vitro Domain Shuffling;

In regard to the 95 clones of L chains selected above, evaluation wascarried out on relative activity of a single L chain clone and relativeactivity of a combination of an L chain clone with H chain of thechimeric antibody.

As a 96-well PS plate for screening, a Black plate (Greiner #655076) forfluorescence measurement was used. First, this PS plate was irradiatedwith oxygen plasma at 30 W for one minute, so that a surface of the PSplate became hydrophilic. Then, each of the top 95 clones of human FabL-PS and Rituxan (registered trademark) Fab H-PS chimera were mixed andarranged so that: a final concentration of urea was 2 M; a finalconcentration of Tween 20 was 1%; and a final concentration of chimericFab H-PS was 25 μg/ml. As a control, a human Fab L-PS library solution(i.e., that does not contain chimeric Fab H-PS) was prepared. This humanFab L-PS library solution was arranged to have a Tween 20 finalconcentration of 0.1% and a urea final concentration of 2 M. Then, 100μl of thus prepared control or thus prepared mixture of a human Fab L-PSclone and Rituxan (registered trademark) Fab H-PS chimera were providedto each well and two-hour incubation was carried out. Then, after the PSplate was washed with 0.1% Tween PBS, 270 μl of 2% BSA-0.1% Tween PBSwas added to each well. Next, the PS plate was subjected to blocking forone hour. Further, after the PS plate was washed with 0.1% Tween PBS, 1μg/ml FITC labeled antigen solution containing 0.1% Tween 20 and 5%human serum was prepared. After 100 μl of thus prepared FITC labeledantigen solution was added to each well, one-hour incubation was carriedout while light was shielded. Subsequently, after the PS plate waswashed with 0.1% Tween PBS, fluorescence intensity was measured by usinga fluorescence plate reader (TECAN infinite M200) (excitationwavelength: 486 nm, fluorescence wavelength: 520 nm).

FIG. 12 shows a result of the measurement. An upper panel of FIG. 12shows fluorescence intensity of combinations of each of the top 95clones of human L chains and H chain of a chimeric antibody. Meanwhile,a lower panel of FIG. 12 shows fluorescence intensity of each singleclone of the top 95 clones of human L chains. As shown in the upperpanel, among combinations of each of the top 95 clones of human L chainsand the chimeric antibody H chain, the fluorescence intensity increasedby 6000 or more in 61 clones (63.8%). In 77 clones (81.9%), thefluorescence intensity increased by 4000 or more. In 93 clones (98.9%),the fluorescence intensity increased by 2000 or more. There was a clonewhose fluorescence intensity increased by 14131 that was a maximumincrease. It became clear that antigen binding ability was significantlyimproved in many of human L chains screened, in a case where each of themany of human L chains screened was combined with H chain derived fromthe chimeric antibody.

(7) Evaluation of Human Fab H-PS Library/Rituxan (Registered Trademark)Chimeric Fab L-PS by Using In Vitro Domain Shuffling (Selection of 94Clones from Among 960 Clones);

Next, by combining L chain of a chimeric antibody and a human H chainlibrary (960 types), a suitable human H chain was screened.

As a 384-well PS plate for screening, a Black plate (BD Falcon #353285)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 27 μl of 1.33% Tween PBS wasadded to each well, 9 μl of the human Fab H-PS library was added to eachwell. Then, 4 μl of Rituxan (registered trademark) chimeric Fab L-PS wasadded to each well. Rituxan (registered trademark) chimeric Fab L-PShere had been prepared so that a concentration of the Rituxan(registered trademark) chimeric Fab H-PS was 250 μg/ml in 2 M urea and 1Tween PBS. Then, two-hour incubation was carried out. After the PS platewas washed with 0.1% Tween PBS, 80 μl of 2% BSA-0.1% Tween PBS was addedto each well. Then, the PS plate was subjected to blocking for one hour.Further, after the PS plate was washed with 0.1% Tween PBS, 1 μg/ml FITClabeled antigen solution was prepared. This FITC labeled antigensolution contained 0.1% Tween 20 and 5% human serum. Then, 40 μl of thusprepared FITC labeled antigen solution was added to each well. Then,while light was shielded, one-hour incubation was carried out.Subsequently, after the PS plate was washed with 0.1% Tween PBS,fluorescence intensity was measured by using a fluorescence plate reader(TECAN infinite M200) (excitation wavelength: 486 nm, fluorescencewavelength: 520 nm).

FIG. 13 shows a result of combining L chain derived from a chimericantibody and a human H chain library (960 types). In FIG. 13, a verticalaxis shows fluorescent intensity, while a horizontal axis shows types ofhuman H chains. In regard to combinations of the L chain and the human Hchain library, the highest fluorescence intensity was 12074 while anaverage fluorescence intensity was 2716. From the human H chains whosefluorescence intensity was 4000 or higher among the 960 clones of thehuman H chains each in combination with the L chain derived from thechimeric antibody, top 94 clones in the order of fluorescence intensitydetected were selected.

(8) Evaluation of Top 94 Clones of Human Fab H-PS Library/Rituxan(Registered Trademark) Chimeric Fab L-PS by Using In Vitro DomainShuffling;

In regard to the 94 clones of H chains selected above, evaluation wascarried out on relative activity of a single H chain clone and specificactivity of a combination of an H chain clone with L chain of a chimericantibody.

As a 96-well PS plate for screening, a Black plate (Greiner #655076) forfluorescence measurement was used. First, this PS plate was irradiatedwith oxygen plasma at 30 W for one minute, so that a surface of the PSplate became hydrophilic. Then, each of the top 94 clones of human FabH-PS library and Rituxan (registered trademark) Fab L-PS chimera eachdissolved in 8 M urea-PBS were mixed and arranged so that: a finalconcentration of urea was 2 M; a final concentration of Tween 20 was 1%;and a final concentration of chimeric Fab L-PS was 25 μg/ml. As acontrol, a human Fab H-PS library solution (i.e., that does not containchimeric Fab L-PS) was prepared. This human Fab H-PS library solutionwas arranged to have a Tween 20 final concentration of 0.1% and a ureafinal concentration of 2 M. Then, 100 μl of thus prepared control orthus prepared mixture of a human Fab H-PS clone and Rituxan (registeredtrademark) Fab L-PS chimera were provided to each well and two-hourincubation was carried out. Then, after the PS plate was washed with0.1% Tween PBS, 270 μl of 2% BSA-0.1% Tween PBS was added to each well.Next, the PS plate was subjected to blocking for one hour. Further,after the PS plate was washed with 0.1% Tween PBS, 1 μg/ml FITC labeledantigen solution containing 0.1% Tween 20 and 5% human serum wasprepared. After 100 μl of thus prepared FITC labeled antigen solutionwas added to each well, one-hour incubation was carried out while lightwas shielded. Subsequently, after the PS plate was washed with 0.1%Tween PBS, fluorescence intensity was measured by using a fluorescenceplate reader (TECAN infinite M200) (excitation wavelength: 486 nm,fluorescence wavelength: 520 nm).

FIG. 14 shows a result of the measurement. An upper panel of FIG. 14shows fluorescence intensity of combinations of each of the top 94clones of human H chain and L chain of a chimeric antibody. Meanwhile, alower panel of FIG. 14 shows fluorescence intensity of each single cloneof the top 94 clones of human H chains. As shown in the upper panel,among combinations of each of the top 94 clones of human H chains andthe chimeric antibody L chain, the fluorescence intensity increased by2000 or more in 17 clones (18.1%). In 83 clones (67.0%), thefluorescence intensity increased by 1000 or more. There was a clonewhose fluorescence intensity increased by 5272 that was a maximumincrease. It became clear that antigen binding ability was significantlyimproved in many of human H chains screened, in a case where each of themany of human H chains screened was combined with L chain derived fromthe chimeric antibody.

(9) Evaluation of Human Fab H-PS library/Human Fab L-PS by Using InVitro Domain Shuffling;

Next, from among the human Fab L-PS and the human Fab H-PS each selectedin the above sections (5) to (8), clones having high antigen bindingability were selected. Here, 5 types of human Fab L-PS (Clone No. 1, 2,3, 11, and 12) and 8 types of human Fab H-PS (Clone No. 1, 2, 3, 4, 11,32, 48, 85, and 91) were selected. Then, each of 5 the types of humanFab L-PS were combined with every one of the 8 types of human Fab H-PS,and antigen binding ability of thus obtained combinations was evaluated.As a control, Rituxan (registered trademark) chimeric Fab L-PS andchimeric Fab H-PS were used. Clone numbers of the human Fab L-PSindicated here are identical to those in FIGS. 11 and 12. Further, Clonenumbers of human Fab H-PS here are identical to those in FIGS. 13 and14. The following explains a specific procedure.

First, as a 96-well PS plate for screening, a Black plate (Greiner#655076) for fluorescence measurement was used. This PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. Then, each of (a) each clone ofhuman Fab H-PS, (b) each clone of human Fab L-PS, (c) Rituxan(registered trademark) chimeric Fab L-PS and (c) Rituxan (registeredtrademark) chimeric Fab H-PS were prepared so that a concentrationbecame 1 mg/l in 8 M urea-PBS-1% Tween 20.

Into each well of a PVC plate, 72 μl of PBS containing 1% Tween 20 and24 μl of PBS containing 8 M urea and 1% Tween 20 were added. Further, 12μl of Fab L-PS and 12 μl of Fab H-PS were added to each well (a totalamount of solution: 120 μl, a final concentration of Fab L-PS and FabH-PS: 100 μg/ml, a final concentration of urea: 2 M, and a finalconcentration of Tween 20:1%). Note that as a control, a sample thatcontains only one of Fab H-PS and Fab L-PS was also prepared. Then, 100μl of each of these was added to each well. Subsequently, the Blackplate was incubated at 25° C. for 2 hours. Then, after the PS plate waswashed with 0.1% Tween PBS, 270 μl of 2% BSA-0.1% Tween PBS was added toeach well. Next, the PS plate was subjected to blocking for one hour.Further, after the PS plate was washed with 0.1% Tween PBS, 1 μg/ml FITClabeled antigen solution containing 0.1% Tween 20 and 5% human serum wasprepared. After 100 μl of thus prepared FITC labeled antigen solutionwas added to each well, one-hour incubation was carried out while lightwas shielded. Subsequently, after the PS plate was washed with 0.1%Tween PBS, fluorescence intensity was measured by using a fluorescenceplate reader (TECAN infinite M200) (excitation wavelength: 486 nm,fluorescence wavelength: 520 nm).

On the PS plate, 40 types in total of human Fab were prepared by usingthe 8 types of human Fab H-PS clones and the 5 types of human Fab L-PSclones. FIG. 15 shows a result of evaluating affinity for FITC labeledantigen. As shown in FIG. 15, there were many combinations of human FabH-PS and human Fab L-PS each of which combinations shows a higher signalas compared to a combination of chimeric Fab H-PS and chimeric Fab L-PSas a control.

FIG. 16 shows a result of calculating specific activity (i.e., signalintensity per unit Fab) obtained by dividing the signal intensityobtained as described above by an amount of Fab H-PS/Fab L-PSimmobilized. As shown in FIG. 16, it was possible to obtain manycombinations of human Fab H-PS and human Fab L-PS each of whichcombinations showed specific activity whose level was equivalent to orhigher than that of the combination of chimeric Fab H-PS chimeric FabL-PS. Therefore, it became clear that a whole human antibody could beproduced from a chimeric antibody by using the present invention.

(10) Evaluation of Human Fab H-PS/Human Fab L-PS Library by Using InVitro Domain Shuffling;

In the present experiment, five types (Clone No. 1, 2, 3, 4, and 11) ofhuman Fab H-PS were selected as clones having high antigen bindingability from among clones of human Fab H-PS selected in the sections (5)to (8) above. With respect to each of thus selected 5 types of human FabH-PS, each of 960 clones of a human Fab L-PS library was combined andaffinity for an antigen was evaluated. The following explains a specificprocedure.

As a 384-well PS plate for screening, a Black plate (BD Falcon #353285)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 27 μl of 1.33% Tween PBS wasadded to each well, 9 μl of the human Fab L-PS library was added to eachwell. Then, 4 μl of each human Fab H-PS clone was added to each well.The human Fab H-PS clone here had been prepared so that a concentrationof the human Fab H-PS clone was 250 μg/ml in 2 M urea and 1% Tween PBS.Then, two-hour incubation was carried out. After the PS plate was washedwith 0.1% Tween PBS, 80 μl of 2% BSA-0.1% Tween PBS was added to eachwell. Then, the PS plate was subjected to blocking for one hour.Further, after the PS plate was washed with 0.1% Tween PBS, 1 μg/ml FITClabeled antigen solution was prepared. This FITC labeled antigensolution contained 0.1% Tween 20 and 5% human serum. Then, 40 μl of thusprepared FITC labeled antigen solution was added to each well. Then,while light was shielded, one-hour incubation was carried out.Ultimately, after the PS plate was washed with 0.1% Tween PBS,fluorescence intensity was measured by using a fluorescence plate reader(TECAN infinite M200) (excitation wavelength: 486 nm, fluorescencewavelength: 520 nm).

(11) Evaluation of Human Fab H-PS Library/Human Fab L-PS by Using InVitro Domain Shuffling;

Similarly, with respect to top five clones (5 types of human Fab L-PS(Clone No. 1, 2, 3, 11 and 12)) in each of which higher signal intensitywas detected in the sections (5) to (8) above, each of 960 clones of ahuman Fab H-PS library was combined and affinity for an antigen wasevaluated. The following explains a specific procedure.

As a 384-well PS plate for screening, a Black plate (BD Falcon #353285)for fluorescence measurement was used. First, this PS plate wasirradiated with oxygen plasma at 30 W for one minute, so that a surfaceof the PS plate became hydrophilic. After 27 μl of 1.33% Tween PBS wasadded to each well, 9 μl of the human Fab H-PS library was added to eachwell. Then, 4 μl of each human Fab L-PS clone was added to each well.The human Fab L-PS clone here had been prepared so that a concentrationof the human Fab L-PS clone was 250 μg/ml in 2 M urea and 1% Tween PBS.Then, two-hour incubation was carried out. After the PS plate was washedwith 0.1% Tween PBS, 80 μl of 2% BSA-0.1% Tween PBS was added to eachwell. Then, the PS plate was subjected to blocking for one hour.Further, after the PS plate was washed with 0.1% Tween PBS, 1 μg/ml FITClabeled antigen solution was prepared. This FITC labeled antigensolution contained 0.1% Tween 20 and 5% human serum. Then, 40 μl of thusprepared FITC labeled antigen solution was added to each well. Then,while light was shielded, one-hour incubation was carried out.Ultimately, after the PS plate was washed with 0.1% Tween PBS,fluorescence intensity was measured by using a fluorescence plate reader(TECAN infinite M200) (excitation wavelength: 486 nm, fluorescencewavelength: 520 nm).

As described above, 5 types of human Fab H-PS clones and 5 types ofhuman Fab L-PS clones were selected. From thus selected human Fab H-PSclones and human Fab L-PS clones, high detection signals were obtainedwhen each of thus selected human Fab H-PS clones and human Fab L-PSclones was mixed with chimeric Fab H-PS or chimeric Fab L-PS. Each ofthus selected five types of human Fab H-PS clones and five types ofhuman Fab L-PS clones was set together thoroughly with every one of 960clones of the human Fab L-PS library or each of 960 clones of the humanFab H-PS library. Thereby, 9600 types of Fab antibodies each made ofhuman Fab H-PS and human Fab L-PS were prepared on a PS plate. In regardto the above human Fab H-PS and human Fab L-PS, affinity for afluorescence labeled antigen was evaluated. FIGS. 17 to 22 show resultsof the evaluation.

More specifically, FIG. 17 shows (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab L-PS librarywith Clone No. 1 of human Fab H-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab L-PSlibrary with Clone No. 2 of human Fab H-PS. Similarly, FIG. 18 shows (a)in an upper panel, a result of Fab antibodies obtained by combining 960types in a human Fab L-PS library with Clone No. 3 of human Fab H-PS and(b) in a lower panel, a result of Fab antibodies obtained by combining960 types in a human Fab L-PS library with Clone No. 4 of human FabH-PS. FIG. 19 shows a result of Fab antibodies obtained by combining 960types in a human Fab L-PS library with Clone No. 11 of human Fab H-PS.

Moreover, FIG. 20 shows (a) in an upper panel, a result of Fabantibodies obtained by combining 960 types in a human Fab H-PS librarywith Clone No. 1 of human Fab L-PS and (b) in a lower panel, a result ofFab antibodies obtained by combining 960 types in a human Fab H-PSlibrary with Clone No. 2 of human Fab L-PS. FIG. 21 shows (a) in anupper panel, a result of Fab antibodies obtained by combining 960 typesin a human Fab H-PS library with Clone No. 3 of human Fab L-PS and (b)in a lower panel, a result of Fab antibodies obtained by combining 960types in a human Fab H-PS library with Clone No. 11 of human Fab L-PS.FIG. 22 shows a result of Fab antibodies obtained by combining 960 typesin a human Fab H-PS library with Clone No. 12 of human Fab L-PS.

As shown in the above drawings, many combinations of human Fab H-PS andhuman Fab L-PS were detected as combinations each having a signal whoselevel was equivalent to or higher than that of a Fab antibody made of acombination of chimeric Fab H-PS and human Fab L-PS or a combination ofhuman Fab H-PS and chimeric Fab L-PS. In particular, it is highly likelythat very high affinity for an antigen is held by the combinations ofhuman Fab H-PS and human Fab L-PS from each of which combinations asignal intensity of 10000 or higher was obtained. Therefore, detailedaffinity evaluation will make it possible to obtain a whole humanantibody clone that has higher affinity and less adverse effect ascompared to a chimeric antibody made of chimeric Fab H-PS and chimericFab L-PS.

INDUSTRIAL APPLICABILITY

The present invention is applicable as a basic technology for antibodyscreening. By obtaining a whole antibody by recloning of a heavy-chainlow-molecular-weight antibody and a light-chain low-molecular-weightantibody each screened as described above, thus obtained whole antibodymay be used, for example, for an antibody drug or a diagnostic product.

The invention claimed is:
 1. An antibody-immobilized carrier comprisingat least two antibody immobilized regions, wherein each of said antibodyimmobilized regions comprises a carrier binding peptide bound to thematerial of a carrier surface and to the C-terminus side of theheavy-chain variable region within a heavy-chain low-molecular-weightantibody, and a separate carrier binding peptide bound to the materialof a carrier surface and the C-terminus side of the light-chain variableregion within a light-chain low-molecular-weight antibody, wherein saidheavy-chain low-molecular-weight antibody and said light-chainlow-molecular-weight antibody within said antibody immobilized regionrecognize different antigens, wherein each of said antibody immobilizedregions are configured such that an antigen may simultaneously bind saidvariable region of said heavy-chain low molecular-weight antibody andsaid variable region of said light-chain low molecular-weight antibodywithin said antibody immobilized region, wherein the material of thecarrier surface is selected from the group consisting of a polystyreneplastic resin, a polycarbonate plastic resin, a polymethyl methacrylateplastic resin, a plastic resin obtained through hydrophilizing apolystyrene plastic resin, a plastic resin obtained throughhydrophilizing a polycarbonate plastic resin, and a plastic resinobtained through hydrophilizing a polymethyl methacrylate plastic resinand wherein the carrier binding peptide is selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQID NO:
 19. 2. The antibody-immobilized carrier as set forth in claim 1,wherein: the carrier binding peptide is a peptide that binds tohydrophilic polystyrene, hydrophilic polycarbonate, or hydrophilicpolymethyl methacrylate.
 3. The antibody-immobilized carrier as setforth in claim 1, wherein: the heavy-chain low-molecular-weight antibodyis a heavy-chain low-molecular-weight antibody consisting of theheavy-chain variable region or a heavy-chain low-molecular-weightantibody consisting of a heavy-chain variable region and a firstheavy-chain constant region.
 4. The antibody-immobilized carrier as setforth in claim 1, comprising at least 10² antibody immobilized regions.5. The antibody-immobilized carrier as set forth in claim 1, wherein theantibody immobilized region includes a region in which the heavy-chainlow-molecular-weight antibody and the light-chain low-molecular-weightantibody form a pair.
 6. The antibody-immobilized carrier as set forthin claim 1, wherein each of said antibody immobilized regions are notidentical.
 7. The antibody-immobilized carrier as set forth in claim 1,wherein said material is a polystyrene plastic resin or a plastic resinobtained through hydrophilizing a polystyrene plastic resin.
 8. Theantibody-immobilized carrier as set forth in claim 1, wherein saidcarrier binding peptide is a polystyrene binding peptide.
 9. Theantibody-immobilized carrier as set forth in claim 8, wherein saidpolystyrene binding peptide comprises SEQ ID NO.
 11. 10. Theantibody-immobilized carrier as set forth in claim 1, wherein thematerial of the carrier surface is a polymethyl methacrylate plasticresin or a plastic resin obtained through hydrophilizing a polymethylmethacrylate plastic resin.
 11. The antibody-immobilized carrier as setforth in claim 10, wherein said carrier binding peptide is a polymethylmethacrylate binding peptide.
 12. The antibody-immobilized carrier asset forth in claim 11, wherein the polymethyl methacrylate bindingpeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO:
 19. 13. Theantibody-immobilized carrier as set forth in claim 1, wherein thematerial of the carrier surface is a polycarbonate plastic resin or aplastic resin obtained through hydrophilizing a polycarbonate plasticresin.
 14. The antibody-immobilized carrier as set forth in claim 13,wherein said carrier binding peptide is a polycarbonate binding peptide.15. The antibody-immobilized carrier as set forth in claim 14, whereinthe polycarbonate binding peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.